Self-degrading enzyme-loaded bio-derived particles

Abstract

The present disclosure provides a composition comprising an enzyme-loaded biologically derived microsphere, which is capable of self-degrading upon rehydration. The invention also provides a method for preparing the enzyme-loaded bio-derived microspheres. The disclosure also provides methods of inducing embolism in a subject using the disclosed microspheres, and methods of treating a disease or disorder in a subject using the disclosed microspheres.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 374,833, filed on September 7, 2022, which is incorporated herein by reference in its entirety. Technical field

[0003] The present disclosure provides compositions comprising self - degrading enzyme - loaded bioderived particles. The present disclosure further provides methods of preparing enzyme - loaded bioderived particles, including methods of using divalent ions and / or photocrosslinking to crosslink the particles. In some aspects, post - treatment of the prepared enzyme - loaded bioderived particles (such as sterilization and dehydration) is used to improve the shelf - life. The present disclosure further provides methods of treating a disease or disorder of a subject using the enzyme - loaded bioderived particles, wherein the disease or disorder would benefit from the administration of these self - degrading particles. The present disclosure further provides methods of using the enzyme - loaded bioderived particles in an embolization procedure in a subject in need thereof. Background art

[0004] Artificial occlusion or embolization of blood vessels in an organ can be used for, for example, (a) controlling bleeding due to trauma, (b) preventing blood from flowing into abnormal blood vessels (such as aneurysms), and / or (c) treating an organ (such as removing a tumor, for transplantation or for surgery). In many cases, permanent embolization of blood vessels is not desired. For such medical interventions, the use of temporary and bioresorbable embolizing agents is desirable. For example, IMP / CS (imipenem / cilastatin) antibiotic particles in the size range of 10 μm to 80 μm have been used as temporary embolizing agents. However, this material may take nearly a month to be completely absorbed (see, for example, Okuno et al.; “Midterm Clinical Outcomes and MR Imaging Changes after Transcatheter Arterial Embolization as a Treatment for Mild to Moderate Radiographic Knee Osteoarthritis Resistant to Conservative Treatment”, J. Vasc. Interv. Radiol. 2017; 28:995 - 1002). Similarly, other embolizing agents have also been used, such as Collagen and thrombin (see, e.g., Vaidya et al.; “An overview of embolic agents”, Semin. Intervent. Radiol. 2008; 25:204-15). However, existing agents have many drawbacks, such as unpredictable resorption rates, lack of agents that selectively degrade the above matrix, and / or migration of embolic agents that cause non-specific occlusion (see, e.g., U.S. Patent Application Publication No. 20130211249). In addition, some embolic agents require processing or preparation steps before their use in the body. For example, Gelfoam must be cut into flakes or slurried. Similarly, the formed autologous blood clot must be collected and reinjected.

[0005] The temporary self-degrading agents have uses beyond embolization and can be used to treat various diseases or disorders in a subject in need thereof. For example, such agents can be used in tissue bulking applications, such as cosmetic fillers and sphincter bulking materials, to provide temporary mechanical support during bone healing, and as organ spacer materials (e.g., SpaceOAR TM , which is used as a temporary spacer between the prostate and the intestine to protect the intestine during radiotherapy for prostate cancer treatment). More generally, the self-degrading agents can be used in degradable implant materials for drug delivery.

[0006] Accordingly, there is a need for self-degrading agents that can exhibit a predictable dissolution rate and methods for preparing these self-degrading agents. In addition, there is a need in the art for methods of using these self-degrading agents to treat diseases or disorders in a subject or to use these self-degrading agents in an embolization procedure in a subject without causing any non-specific occlusion in the body.

[0007] Incorporation by reference

[0008] All publications, patents, and patent applications herein are incorporated herein 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. In the event of a conflict between the terms herein and the terms of the incorporated references, the terms herein shall control. SUMMARY OF THE INVENTION

[0009] In one aspect, the present disclosure provides microspheres capable of self-degrading upon rehydration, comprising: an enzyme pretreated by varying temperature, by varying pH, and / or with a metal ion enzyme inhibitor; and a crosslinked biomaterial; wherein: the crosslinked biomaterial forms a bio-derived microsphere encapsulating the enzyme; and

[0010] The microspheres are substantially free of water and / or are sterilized. In one embodiment, at least one of (i)-(iv) applies: (i) the enzyme is an enzyme that acts on and degrades a biomaterial; (ii) the biomaterial comprises a polysaccharide, protein, or glycoprotein; (iii) the microspheres further comprise a photoinitiator and the biomaterial comprises a photocrosslinkable moiety that is photocrosslinked; or (iv) the biomaterial is crosslinked by divalent metal ions. In one embodiment, the self-degradation of the microspheres is controlled by one or more of the following: pretreatment of the enzyme, the concentration of the enzyme in the microspheres, enzyme activity, the predetermined molecular weight of the biomaterial, the divalent metal ions used to crosslink the biomaterial, and the amount of divalent metal ions used to crosslink the biomaterial. In one embodiment, the enzyme activity is between about 0.0075 U / mg and 0.25 U / mg of biomaterial and the microspheres degrade over a period of greater than about 20 minutes to less than about 4 hours; the enzyme activity is between about 0.005 U / mg and about ≥0.0025 U / mg of biomaterial and the microspheres degrade over a period of greater than about 5 days to less than about 30 days; or the enzyme activity is less than about 0.0025 U / mg of biomaterial and the microspheres degrade over a period of greater than about 30 days. In one embodiment, at least one of (i)-(iv) applies: (i) the residual water content of the microspheres is between about 1% mass and about 10% mass; (ii) the microspheres are lyophilized or dehydrated using supercritical CO2; (iii) the microspheres are sterilized with about 6-10 kGy of gamma radiation; or (iv) the microspheres further comprise an anti-inflammatory agent, chemotherapeutic agent, antioxidant, corticosteroid, or a combination thereof. In one embodiment, the biomaterial comprises alginate and the enzyme is alginate lyase, the biomaterial comprises pectin and the enzyme is pectinase, the biomaterial comprises hyaluronic acid and the enzyme is hyaluronidase, the biomaterial comprises gelatin and the enzyme is matrix metalloproteinase or protease, the biomaterial comprises albumin and the enzyme is peptidase, the biomaterial comprises collagen and the enzyme is protease, the biomaterial comprises fibrinogen and the enzyme is plasmin, the biomaterial comprises silk fibroin and the enzyme is protease, the biomaterial comprises starch and the enzyme is amylase, the biomaterial comprises chitosan and the enzyme is chitosanase or lysozyme, the biomaterial comprises agar / agarose and the enzyme is agarase, the biomaterial comprises carrageenan and the enzyme is carrageenase, the biomaterial comprises pullulan and the enzyme is pullulanase, the biomaterial comprises dextran and the enzyme is dextranase, the biomaterial comprises b-glycan and the enzyme is b-glycanase, the biomaterial comprises cellulose and the enzyme is cellulase, or the biomaterial comprises lignin and the enzyme is ligninase.

[0011] In another aspect, the present disclosure provides a method for preparing microspheres capable of self-degrading upon rehydration, the method comprising: forming droplets from a precursor solution comprising: (i) an enzyme pretreated by varying temperature, by varying pH, and / or with a metal ion enzyme inhibitor; and (ii) a biomaterial; contacting the droplets with a gelling bath comprising a cryoprotectant and a divalent metal ion, thereby crosslinking the biomaterial to form bio-derived microspheres encapsulating the enzyme; and dehydrating and optionally sterilizing the microspheres, thereby substantially removing water from the microspheres. In one embodiment, at least one of (i)-(v) applies: (i) the precursor solution further comprises one or more cryoprotectants; (ii) the enzyme is an enzyme that acts on the biomaterial and degrades the biomaterial; (iii) the biomaterial comprises a polysaccharide, a protein, or a glycoprotein; (iv) the microspheres comprise: alginate particles encapsulating alginate lyase, pectin particles encapsulating pectinase, gelatin particles encapsulating matrix metalloproteinase or protease, or carrageenan particles encapsulating carrageenase; or (v) the microspheres self-degrade in less than about 4 hours, in a time period greater than about 2 days to less than about 5 days, in a time period greater than about 5 days to less than about 30 days, or in greater than about 30 days. In one embodiment, the self-degradation of the microspheres is controlled by one or more of: pretreatment of the enzyme, the concentration of the enzyme in the microspheres, enzyme activity, the predetermined molecular weight of the biomaterial, the divalent metal ion used to crosslink the biomaterial, and the amount of the divalent metal ion used to crosslink the biomaterial. In one embodiment, the droplets are contacted with the gelling bath in the range of 10 minutes to 1 hour, and the resulting microspheres degrade in a time period greater than about 20 minutes to less than about 4 hours; the droplets are contacted with the gelling bath in the range of 1 hour to 12 hours, and the resulting microspheres degrade in a time period greater than about 5 days to less than about 30 days; or the droplets are contacted with the gelling bath in the range of 12 hours to 24 hours, and the resulting microspheres degrade in a time period greater than about 30 days. In one embodiment, at least one of (i)-(iv) applies: (i) the residual water content of the microspheres is between about 1% mass and about 10% mass; (ii) dehydration comprises freeze-drying the microspheres or drying the microspheres using supercritical CO2; (iii) sterilization comprises irradiating the microspheres with γ-radiation at 6-10 kGy; or (iv) the precursor solution and / or the gelling bath further comprises an anti-inflammatory agent, a chemotherapeutic agent, an antioxidant, or a combination thereof.

[0012] In yet another aspect, the present disclosure provides a method for preparing photopolymerizable microspheres that are capable of self-degrading upon rehydration, the method comprising: forming droplets from a precursor solution comprising: (i) an enzyme pretreated by varying temperature, by varying pH, and / or with a metal ion enzyme inhibitor; (ii) a biomaterial comprising a photocrosslinkable moiety; (iii) a photoinitiator; irradiating the droplets to crosslink the biomaterial to form photopolymerized bio-derived microspheres encapsulating the enzyme; and dehydrating and optionally sterilizing the microspheres to substantially remove water from the microspheres. In one embodiment, at least one of (i)-(v) applies: (i) the photocrosslinkable moiety is selected from acrylate groups, methacrylate groups, vinyl groups, and allyl groups; (ii) the precursor solution further comprises one or more cryoprotectants; (iii) the enzyme is an enzyme that acts on the biomaterial and degrades the biomaterial; (iv) the biomaterial comprises a polysaccharide, a protein, or a glycoprotein; or (v) the microspheres self-degrade in less than about 4 hours, in a time period greater than about 2 days to less than about 5 days, in a time period greater than about 5 days to less than about 30 days, or in greater than about 30 days. In one embodiment, the microspheres comprise alginate particles encapsulating alginate lyase, pectin particles encapsulating pectinase, hyaluronic acid particles encapsulating hyaluronidase, gelatin particles encapsulating matrix metalloproteinase or protease, albumin particles encapsulating peptidase, collagen particles encapsulating protease, fibrinogen particles encapsulating plasmin, silk fibroin particles encapsulating protease, starch particles encapsulating amylase, chitosan particles encapsulating chitosanase or lysozyme, agar / agarose particles encapsulating agarase, carrageenan particles encapsulating carrageenase, pullulan particles encapsulating pullulanase, dextran particles encapsulating dextranase, b-glycan particles encapsulating b-glycanase, cellulose particles encapsulating cellulase, or lignin particles encapsulating ligninase. In one embodiment, the self-degradation of the microspheres is controlled by one or more of the following: pretreatment of the enzyme, the concentration of the enzyme in the microspheres, enzyme activity, the predetermined molecular weight of the biomaterial, and the amount of time the droplets are irradiated. In one embodiment, at least one of (i)-(iv) applies: (i) the residual water content of the microspheres is between about 1% mass and about 10% mass; (ii) dehydration comprises freeze-drying the microspheres or drying the microspheres using supercritical CO2; (iii) sterilization comprises irradiating the microspheres with 6-10 kGy of gamma radiation; or (iv) the precursor solution further comprises an anti-inflammatory agent, a chemotherapeutic agent, an antioxidant, or a combination thereof.

[0013] In yet another aspect, the present disclosure provides a method for preparing microspheres that are capable of self-degrading upon rehydration, the method comprising: forming droplets from a precursor solution comprising: (i) a biomaterial comprising covalently crosslinkable moieties; and (ii) a homobifunctional crosslinker or a heterobifunctional crosslinker; covalently crosslinking the biomaterial to form bio-derived microspheres; swelling an enzyme that has been pretreated by varying temperature, by varying pH, and / or with a metal ion enzyme inhibitor into the microspheres such that the bio-derived microspheres encapsulate the enzyme; and dehydrating the microspheres and optionally sterilizing them to substantially remove water from the microspheres. In one embodiment, at least one of (i)-(v) applies: (i) the covalently crosslinkable moieties comprise amino groups or carboxyl groups; (ii) the precursor solution further comprises one or more cryoprotectants; (iii) the enzyme is an enzyme that acts on and degrades the biomaterial; (iv) the biomaterial comprises a polysaccharide, a protein, or a glycoprotein; or (v) the microspheres self-degrade in less than about 4 hours, in a time period greater than about 2 days to less than about 5 days, in a time period greater than about 5 days to less than about 30 days, or in greater than about 30 days. In one embodiment, the microspheres comprise alginate particles encapsulating alginate lyase, pectin particles encapsulating pectinase, hyaluronic acid particles encapsulating hyaluronidase, gelatin particles encapsulating matrix metalloproteinase or protease, albumin particles encapsulating peptidase, collagen particles encapsulating protease, fibrinogen particles encapsulating plasmin, silk fibroin particles encapsulating protease, starch particles encapsulating amylase, chitosan particles encapsulating chitosanase or lysozyme, agar / agarose particles encapsulating agarase, carrageenan particles encapsulating carrageenase, pullulan particles encapsulating pullulanase, dextran particles encapsulating dextranase, β-glycan particles encapsulating β-glycanase, cellulose particles encapsulating cellulase, or lignin particles encapsulating ligninase. In one embodiment, the self-degradation of the microspheres is controlled by one or more of the following: pretreatment of the enzyme, concentration of the enzyme in the microspheres, enzyme activity, predetermined molecular weight of the biomaterial, and the homobifunctional crosslinker or heterobifunctional crosslinker used to crosslink the biomaterial. In one embodiment, at least one of (i)-(iv) applies: (i) the residual water content of the microspheres is between about 1% mass and about 10% mass; (ii) dehydration comprises freeze-drying the microspheres or drying the microspheres using supercritical CO2; (iii) sterilization comprises irradiating the microspheres with γ-radiation at 6-10 kGy; or (iv) the precursor solution further comprises an anti-inflammatory agent, a chemotherapeutic agent, an antioxidant, or a combination thereof.

[0014] In yet another aspect, the present disclosure provides a method for preparing microspheres that are capable of self-degrading upon rehydration, the method comprising: forming droplets from a precursor solution comprising: (i) a biomaterial comprising a covalently crosslinkable moiety; (ii) an enzyme that has been pretreated by varying temperature, by varying pH, and / or with a metal ion enzyme inhibitor; and (iii) a homobifunctional crosslinker or a heterobifunctional crosslinker; covalently crosslinking the biomaterial to form a bio-derived microsphere encapsulating the enzyme; and dehydrating and optionally sterilizing the microsphere to substantially remove water from the microsphere. In one embodiment, at least one of (i)-(v) applies: (i) the covalently crosslinkable moiety comprises an amino group or a carboxyl group; (ii) the precursor solution further comprises one or more cryoprotectants; (iii) the enzyme is an enzyme that acts on and degrades the biomaterial; (iv) the biomaterial comprises a polysaccharide, a protein, or a glycoprotein; or (v) the microsphere self-degrades in less than about 4 hours, in a time period greater than about 2 days to less than about 5 days, in a time period greater than about 5 days to less than about 30 days, or in greater than about 30 days. In one embodiment, the microsphere comprises alginate particles encapsulating alginate lyase, pectin particles encapsulating pectinase, hyaluronic acid particles encapsulating hyaluronidase, gelatin particles encapsulating matrix metalloproteinase or protease, albumin particles encapsulating peptidase, collagen particles encapsulating protease, fibrinogen particles encapsulating plasmin, silk fibroin particles encapsulating protease, starch particles encapsulating amylase, chitosan particles encapsulating chitosanase or lysozyme, agar / agarose particles encapsulating agarase, carrageenan particles encapsulating carrageenase, pullulan particles encapsulating pullulanase, dextran particles encapsulating dextranase, β-glycan particles encapsulating β-glycanase, cellulose particles encapsulating cellulase, or lignin particles encapsulating ligninase. In one embodiment, the self-degradation of the microsphere is controlled by one or more of: pretreatment of the enzyme, concentration of the enzyme in the microsphere, enzyme activity, predetermined molecular weight of the biomaterial, and the homobifunctional crosslinker or heterobifunctional crosslinker used to crosslink the biomaterial. In one embodiment, at least one of (i)-(iv) applies: (i) the residual water content of the microsphere is between about 1% mass and about 10% mass; (ii) dehydration comprises freeze-drying the microsphere or drying the microsphere using supercritical CO2; (iii) sterilization comprises irradiating the microsphere with γ-radiation at 6-10 kGy; or (iv) the precursor solution further comprises an anti-inflammatory agent, a chemotherapeutic agent, an antioxidant, or a combination thereof.

[0015] In yet another aspect, the present disclosure provides a method for preparing thermogelating microspheres capable of self-degrading upon rehydration, the method comprising: heating a precursor solution comprising a biomaterial to melt the biomaterial; adding an enzyme pretreated by varying temperature, by varying pH, and / or with a metal ion enzyme inhibitor to the precursor solution; forming droplets from the precursor solution; cooling the droplets to form thermogelating bioderived microspheres encapsulating the enzyme; and dehydrating and optionally sterilizing the microspheres to substantially remove water from the microspheres. In one embodiment, at least one of (i)-(iv) applies: (i) the precursor solution further comprises one or more cryoprotectants; (ii) the enzyme is an enzyme that acts on and degrades the biomaterial; (iii) the biomaterial comprises a polysaccharide, a protein, or a glycoprotein; or (iv) the microspheres self-degrade in less than about 4 hours, in a time period greater than about 2 days to less than about 5 days, in a time period greater than about 5 days to less than about 30 days, or in greater than about 30 days. In one embodiment, the microspheres comprise pectin particles encapsulating pectinase, gelatin particles encapsulating matrix metalloproteinase or protease, albumin particles encapsulating peptidase, collagen particles encapsulating protease, fibrinogen particles encapsulating plasmin, silk fibroin particles encapsulating protease, starch particles encapsulating amylase, chitosan particles encapsulating chitosanase or lysozyme, or agar / agarose particles encapsulating agarase. In one embodiment, the self-degradation of the microspheres is controlled by one or more of the following: pretreatment of the enzyme, concentration of the enzyme in the microspheres, enzyme activity, and predetermined molecular weight of the biomaterial. In one embodiment, at least one of (i)-(iv) applies: (i) the residual water content of the microspheres is between about 1% mass and about 10% mass; (ii) dehydration comprises freeze-drying the microspheres or drying the microspheres using supercritical CO2; (iii) sterilization comprises irradiating the microspheres with γ-radiation at 6-10 kGy; or (iv) the precursor solution further comprises an anti-inflammatory agent, a chemotherapeutic agent, an antioxidant, or a combination thereof.

[0016] In yet another aspect, the present disclosure provides a method for preparing microspheres capable of self-degrading, the method comprising: forming a precursor solution comprising: (i) an enzyme; and (ii) a biomaterial; passing the precursor solution through a needle under the influence of an electrostatic potential to form droplets; and contacting the droplets with a gelling bath comprising divalent metal ions, thereby crosslinking the biomaterial to form bio-derived microspheres encapsulating the enzyme. In one embodiment, the method further comprises dehydrating the microspheres and optionally sterilizing them, thereby substantially removing water from the microspheres to form microspheres capable of self-degrading upon rehydration. In one embodiment, at least one of (i)-(v) applies: (i) the precursor solution further comprises one or more cryoprotectants; (ii) the enzyme is an enzyme that acts on the biomaterial and degrades the biomaterial; (iii) the biomaterial comprises a polysaccharide, protein or glycoprotein; (iv) the microspheres self-degrade in less than about 4 hours, in a time period greater than about 2 days to less than about 5 days, in a time period greater than about 5 days to less than about 30 days or in greater than about 30 days; or (v) the self-degradation of the microspheres is controlled by one or more of: pretreatment of the enzyme, concentration of the enzyme in the microspheres, enzyme activity, predetermined molecular weight of the biomaterial, divalent metal ions used to crosslink the biomaterial, and amount of divalent metal ions used to crosslink the biomaterial. In one embodiment, the microspheres comprise: alginate particles encapsulating alginate lyase, pectin particles encapsulating pectinase, hyaluronic acid particles encapsulating hyaluronidase, gelatin particles encapsulating matrix metalloproteinase or protease, albumin particles encapsulating peptidase, collagen particles encapsulating protease, fibrinogen particles encapsulating plasmin, silk fibroin particles encapsulating protease, starch particles encapsulating amylase, chitosan particles encapsulating chitosanase or lysozyme, agar / agarose particles encapsulating agarase, carrageenan particles encapsulating carrageenase, pullulan particles encapsulating pullulanase, dextran particles encapsulating dextranase, b-glycan particles encapsulating b-glycanase, cellulose particles encapsulating cellulase or lignin particles encapsulating ligninase. In one embodiment, at least one of (i)-(iv) applies: (i) the residual water content of the microspheres is between about 1% mass and about 10% mass; (ii) dehydration comprises freeze-drying the microspheres or drying the microspheres using supercritical CO2; (iii) sterilization comprises irradiating the microspheres with γ-radiation at 6-10 kGy; or (iv) the precursor solution and / or the gelling bath further comprises an anti-inflammatory agent, a chemotherapeutic agent, an antioxidant or a combination thereof.

[0017] In yet another aspect, the present disclosure provides a method for inducing self-degrading embolization in a subject in need thereof, comprising administering a plurality of the above-described microspheres into a blood vessel of the subject. In one embodiment, the blood vessel is the geniculate artery and / or the method induces prostatic artery embolization, induces uterine artery embolization, or the microspheres comprise a chemotherapeutic agent or are mixed with a chemotherapeutic agent, and the method induces transarterial chemoembolization (TACE).

[0018] In yet another aspect, the present disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a plurality of the above-described microspheres. In one embodiment, the disease or disorder is selected from tendinopathy, osteoarthritis, frozen shoulder, tennis elbow (lateral epicondylitis), golfer's elbow (medial epicondylopathy), pitcher's elbow (flexor tendinitis), Achilles tendinopathy, plantar fasciitis, symptomatic accessory navicular, hamstring tendinopathy, jumper's knee (patellar tendinitis), runner's knee (patellofemoral pain syndrome (PFPS)), pes anserine bursitis (knee pain), posterior tibial tendon disease, wrist (TFCC - triangular fibrocartilage complex) tendon disease, trigger finger (stenosing flexor tenosynovitis), and hemarthrosis.

[0019] In yet another aspect, the present disclosure provides a method of rapidly degrading microspheres in a subject, comprising administering to the subject a bail out solution, wherein a plurality of the above-described microspheres have been previously administered to the subject, and the bail out solution comprises an enzyme capable of degrading the microspheres. In one embodiment, the enzyme is complementary to the biomaterial used to form the plurality of microspheres. In one embodiment, the microspheres comprise alginate particles encapsulating alginate lyase and the enzyme is alginate lyase, the microspheres comprise pectin particles encapsulating pectinase and the enzyme is pectinase, the microspheres comprise hyaluronic acid particles encapsulating hyaluronidase and the enzyme is hyaluronidase, the microspheres comprise gelatin particles encapsulating matrix metalloproteinase or protease and the enzyme is matrix metalloproteinase or protease, the microspheres comprise albumin particles encapsulating peptidase and the enzyme is peptidase, the microspheres comprise collagen particles encapsulating protease and the enzyme is protease, the microspheres comprise fibrinogen particles encapsulating plasmin and the enzyme is plasmin, the microspheres comprise fibroin particles encapsulating protease and the enzyme is protease, the microspheres comprise starch particles encapsulating amylase and the enzyme is amylase, the microspheres comprise chitosan particles encapsulating chitosanase or lysozyme and the enzyme is chitosanase or lysozyme, the microspheres comprise agar / agarose particles encapsulating agarase and the enzyme is agarase, the microspheres comprise carrageenan particles encapsulating carrageenase and the enzyme is carrageenase, the microspheres comprise pullulan particles encapsulating pullulanase and the enzyme is pullulanase, the microspheres comprise dextran particles encapsulating dextranase and the enzyme is dextranase, the microspheres comprise β - glycan particles encapsulating β - glycanase and the enzyme is β - glycanase, the microspheres comprise cellulose particles encapsulating cellulase and the enzyme is cellulase, or the microspheres comprise lignin particles encapsulating ligninase and the enzyme is ligninase. In one embodiment, the bail out solution further comprises a divalent metal chelator.

[0020] In yet another aspect, the present disclosure provides a method for rapidly degrading divalent metal ion-crosslinked microspheres in a subject, comprising administering an emergency rescue solution to the subject, wherein a plurality of the above divalent metal ion-crosslinked microspheres have been previously administered to the subject, and the emergency rescue solution comprises an anion, a phosphate buffer, or a combination thereof. In one embodiment, at least one of (i)-(iii) applies: (i) the anion comprises citrate; (ii) the phosphate buffer comprises phosphate buffered saline; and (iii) the microspheres comprise alginate particles encapsulating alginate lyase, pectin particles encapsulating pectinase, or carrageenan particles encapsulating carrageenase. In one embodiment, the microspheres comprise alginate particles encapsulating alginate lyase, pectin particles encapsulating pectinase, or carrageenan particles encapsulating carrageenase; and wherein the anion comprises citrate; or the microspheres comprise alginate particles encapsulating alginate lyase, pectin particles encapsulating pectinase, or carrageenan particles encapsulating carrageenase; and wherein the phosphate buffer comprises phosphate buffered saline.

[0021] In yet another aspect, the present disclosure provides a kit comprising: (i) a plurality of the above-described microspheres; and an enzyme capable of rapidly degrading the microspheres when dissolved to form a solution; or (ii) a plurality of the above-described divalent metal ion cross-linked microspheres; and an inorganic salt capable of rapidly degrading the microspheres when dissolved to form a solution. In one embodiment, the enzyme of (i) is complementary to the biomaterial used to form the plurality of microspheres. In one embodiment, for the plurality of microspheres of (i): the microspheres comprise alginate particles encapsulating alginate lyase and the enzyme is alginate lyase, the microspheres comprise pectin particles encapsulating pectinase and the enzyme is pectinase, the microspheres comprise hyaluronic acid particles encapsulating hyaluronidase and the enzyme is hyaluronidase, the microspheres comprise gelatin particles encapsulating matrix metalloproteinase or protease and the enzyme is matrix metalloproteinase or protease, the microspheres comprise albumin particles encapsulating peptidase and the enzyme is peptidase, the microspheres comprise collagen particles encapsulating protease and the enzyme is protease, the microspheres comprise fibrinogen particles encapsulating plasmin and the enzyme is plasmin, the microspheres comprise fibroin particles encapsulating protease and the enzyme is protease, the microspheres comprise starch particles encapsulating amylase and the enzyme is amylase, the microspheres comprise chitosan particles encapsulating chitosanase or lysozyme and the enzyme is chitosanase or lysozyme, the microspheres comprise agar / agarose particles encapsulating agarase and the enzyme is agarase, the microspheres comprise carrageenan particles encapsulating carrageenase and the enzyme is carrageenase, the microspheres comprise pullulan particles encapsulating pullulanase and the enzyme is pullulanase, the microspheres comprise dextran particles encapsulating dextranase and the enzyme is dextranase, the microspheres comprise β-glycan particles encapsulating β-glycanase and the enzyme is β-glycanase, the microspheres comprise cellulose particles encapsulating cellulase and the enzyme is cellulase, or the microspheres comprise lignin particles encapsulating ligninase and the enzyme is ligninase. In one embodiment, for the plurality of microspheres of (ii): the microspheres comprise alginate particles encapsulating alginate lyase, pectin particles encapsulating pectinase, or carrageenan particles encapsulating carrageenase. In one embodiment, the inorganic salt of (ii) releases citrate or phosphate when dissolved to form a solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The foregoing Summary, as well as the following detailed description of embodiments of the compositions and fluid delivery devices, will be better understood when read in conjunction with the accompanying drawings of the exemplary embodiments. However, it should be understood that the embodiments of the present disclosure are not limited to the precise arrangements and instrumentalities shown.

[0023] Figure 1 Describe the general procedure for preparing a dehydrated and sterile composition of the bio-derived microspheres of the present disclosure that contain complementary enzymes.

[0024] Figure 2A Describe the preparation and property customization of the exemplary microspheres of the present disclosure. Dissolve alginate and alginate lyase in an aqueous medium. The microspheres prepared by a conventional method are gelled by cation cross-linking of the alginate + lyase droplets.

[0025] Figure 2B Describe the preparation of exemplary microspheres of the present disclosure and the customization of their properties. The microspheres are lyophilized with an optional cryoprotectant to remove water and "freeze" enzyme activity, preventing premature degradation during storage. The microspheres are sterilized in this form.

[0026] Figure 2C Describe the preparation of exemplary microspheres of the present disclosure and the customization of their properties. Degradation properties can be controlled by varying lyase and alginate parameters and preparation conditions to produce particles with degradation rates varying from days to months, depending on the indication to be treated.

[0027] Figure 3A Describe the post-preparation processes and methods of use of the exemplary particles. Lyophilized alginate particles are prepared to remove water and freeze enzyme activity. The cryoprotectant protects the enzyme and the microsphere structure to allow for shape recovery upon hydration.

[0028] Figure 3B Describe the post-preparation processes and methods of use of the exemplary particles. The particles are reconstituted in an aqueous medium during use, hydrating the particles and rendering the lyase catalytically active.

[0029] Figure 3C Describe the post-preparation processes and methods of use of the exemplary particles. The particles are prepared in a suitable suspension for intra-arterial delivery for a specified embolization procedure (e.g., uterine fibroid embolization). When in the body, enzyme activity is enhanced and alginate chains are cleaved, releasing cations, polymer chain fragments, and lyase into the body where they can be reabsorbed or excreted.

[0030] Figure 4A Describe enzyme concentration-dependent alginate particle degradation. A line graph is provided showing the degradation of alginate particles over time as a function of enzyme concentration.

[0031] Figure 4B Describe enzyme concentration-dependent alginate particle degradation. Images of the particles after the degradation period and samples with different enzyme concentrations.

[0032] Figure 5 Describe Ca prepared from alginate 2+ Enzyme concentration-dependent degradation of Ca-crosslinked alginate microspheres (viscosity 144 cps, 1% w / v alginate, 25 °C). Alginate lyase precursor solutions containing 0.25 U / ml, 0.5 U / ml, and 1 U / ml alginate lyase and 1.5% w / v alginate. Control microspheres are enzyme-free. Scale bar = 5 mm.

[0033] Figure 6 Describe pH-dependent regulation of enzyme conformation / activity.

[0034] Figure 7Description of the preparation of Ca - crosslinked alginate microspheres loaded with 5 U of alginate lyase using an alginate lyase - alginate precursor solution pretreated with (a) 0.1 M acetate buffer (pH 4.0) and (b) 0.01 M phosphate buffer (pH 6.5). 2+ Crosslinked alginate microspheres.

[0035] Figure 8 Description of the microscopic images of the degraded Ca - crosslinked alginate microspheres prepared from an alginate lyase (AL) - alginate (Alg) precursor solution containing 5 U of the AL enzyme, where the precursor solution was pretreated with (a and c) 0.1 M acetate buffer (pH 4.0) and (b and d) 0.01 M phosphate buffer (pH 6.5) in phosphate buffer at 0 and 72 hours. (e) Absorbance spectra of the degradation products of the alginate - AL microspheres corresponding to the (c) and (d) samples. 2+ Description of the absorbance spectra of the degradation products obtained from an alginate - alginate lyase (AL, 5 U) precursor solution (alginate - AL A.B) in acetate buffer (A.B) (pH 4), an alginate - alginate lyase (AL) precursor solution (alginate - AL P.B) in 0.01 M phosphate buffer (P.B) (pH 6.5), and an alginate lyase ((alginate(P.B) - AL(A.B)) pre - incubated in A.B for 15 minutes and mixed with alginate dissolved in 0.01 M P.B for 30 minutes, respectively, at 1 - 4 °C and 37 °C.

[0036] Figure 9 Description of the in - vitro degradation study of calcium - complexed alginate particles loaded with alginate lyase in a liver model at 0 hours.

[0037] Figure 10A Description of the in - vitro degradation study of calcium - complexed alginate particles loaded with alginate lyase in a liver model at 24 hours.

[0038] Figure 10B Description of the in - vitro degradation study of calcium - complexed alginate particles loaded with alginate lyase in a liver model at 48 hours.

[0039] Figure 10C Description of the Ca - crosslinked alginate microspheres loaded with 1 U of alginate lyase and 0.5% w / v PVP 40KDa + 0.5% w / v trehalose (A and A`) and 0.5% w / v hydroxypropyl - β - cyclodextrin (B and B`), respectively, before and after lyophilization.

[0040] Figure 11 Description of the Ca - crosslinked alginate microspheres loaded with 1 U of alginate lyase and 0.5% w / v PVP 40KDa + 0.5% w / v trehalose (A and A`) and 0.5% w / v hydroxypropyl - β - cyclodextrin (B and B`), respectively, before and after lyophilization. 2+ Crosslinked alginate microspheres.

[0041] Figure 12Description of freeze-dried Ca 2+ Microscopic images of cross-linked alginate microspheres loaded with 5 U of alginate lyase and (a) 0.5% w / v PVP 40 kDa + 0.5% w / v trehalose and (b) 0.5% w / v hydroxypropyl-β-cyclodextrin (before degradation); (c) and (d) samples corresponding to (a) and (b), respectively, after incubation at 37 °C for 72 h and degradation in 0.01 M phosphate buffer (pH 6.5). Absorbance spectra (e) of the degradation products of samples (c) and (d) in 0.01 M phosphate buffer (pH 6.5) after incubation at 37 °C for 72 h.

[0042] Figure 13 Demonstrate the in vitro biocompatibility of calcium alginate particles loaded with alginate lyase.

[0043] Figure 14 Demonstrate the resorbable beads after freeze-drying and rehydration in saline under physiological conditions observed at 4 time points.

[0044] Figure 15 Is a graph showing the re-establishment of flow after embolization and subsequent degradation of rehydrated resorbable alginate beads containing 0.05 U of alginate lyase in the Elastrat liver model.

[0045] Figure 16 Is a graph showing the re-establishment of flow after embolization and subsequent degradation of rehydrated resorbable alginate beads and permanent beads containing 0.01 U (without (w / o) additional cross-linking in CaCl2) and with additional cross-linking (0.01 U + CaCl2) in CaCl2 solution in the Elastrat liver model.

[0046] Figure 17 Is a graph showing alkaline pH-dependent reversible alginate lyase activity.

[0047] Figure 18 Is a graph showing the effect of electron beam sterilization on the activity of alginate lyase (0.05 U) encapsulated in Ca 2+ Cross-linked alginate beads (beads without enzyme are shown as control).

[0048] Figure 19 Describe an exemplary syringe that shows compartments for the suspension medium and dry alginate microspheres. By applying pressure on the plunger, the separation membrane may be torn inside the syringe, thereby reconstituting the dry alginate microspheres in the suspension medium containing calcium chloride solution.

[0049] Figure 20A - 20C Depict the degradation of calcium-crosslinked pectin beads encapsulating pectin lyase after 24 h. Figure 20AIs an image of degraded pectin lyase - pectin beads. Figure 20B Is an image of control calcium - crosslinked pectin beads (without lyase). Figure 20C Is a bar graph of the absorbance of calcium - crosslinked pectin lyase - pectin beads and pectin - without lyase pectin beads.

[0050] Figure 21A - 21B Illustrates the enzyme - concentration - dependent degradation of Ca 2+ Crosslinked alginate microspheres prepared from alginate (viscosity 160 cps, 2% w / v alginate, 25 °C). The alginate lyase precursor solution contains 0.15 U / mL, 0.5 U / mL, and 1 U / mL alginate lyase. Control (permanent) microspheres do not contain enzyme.

[0051] Figure 22A - 22D Provides an image of a pig kidney that has been injected with a reversible embolization fluid. Figure 22A Is an image of a renal segment immediately after injection of 1.8 mL of alginate microspheres (embolization fluid) that do not encapsulate alginate lyase. Figure 22B Shows partial vascular recanalization of flowing vessels within 10 minutes after injection of an alginate lyase rescue solution. Figure 22C Shows an image in which, 2 hours after occlusion, an alginate lyase rescue solution is injected to reverse the occlusion and restore the original flow. Figure 22D Shows complete vascular recanalization of a pig kidney model 24 hours later.

[0052] Figure 23A - 23E Shows the reperfusion of a pig kidney embolized with resorbable alginate microspheres loaded with 0.15 U and 0.0875 U of alginate lyase. Figure 23A - 23D Shows the opening of blood vessels by alginate microspheres loaded with 0.15 U and 0.0875 U of alginate lyase within 20 hours. Figure 23E Shows the parenchyma of the embolized site of the kidney. Detailed Description

[0053] Overview

[0054] Generally, the present disclosure provides dehydrated and / or sterilized compositions that comprise bioderived particles, and the bioderived particles contain enzymes that can act on the bioderived particles. In one embodiment, the enzyme that can act on the bioderived particles is its complementary enzyme. In one embodiment, the enzyme acts to control the degradation of the bioderived particles. In one embodiment, the bioderived particles are microparticles.

[0055] In one embodiment, the bio-derived particles comprise a protein or glycoprotein biomaterial. Exemplary bio-derived particles comprising a protein or glycoprotein include, but are not limited to, albumin particles, collagen particles, gelatin particles, fibrinogen particles, or fibroin particles, each particle containing an enzyme that can act on the bio-derived particle. In one embodiment, the bio-derived particle-enzyme combinations are albumin containing peptidase, collagen or gelatin containing protease, gelatin containing matrix metalloproteinase, fibrinogen containing plasmin, or fibroin containing protease. In another embodiment, the bio-derived particles comprise a polysaccharide. Exemplary bio-derived particles comprising a polysaccharide include, but are not limited to, alginate particles, hyaluronic acid particles, pectin particles, starch particles, chitosan particles, agar / agarose particles, carrageenan particles, pullulan particles, dextran particles, b-glycan particles, cellulose particles, or lignin particles, each particle containing an enzyme that can act on the bio-derived particle. In one embodiment, the bio-derived particle-enzyme combinations are alginate containing alginase (alginate lyase), hyaluronic acid containing hyaluronidase, pectin containing pectinase, starch containing amylase, chitosan containing chitosanase or lysozyme, agar / agarose containing agarase, carrageenan containing carrageenase, pullulan containing pullulanase, dextran containing dextranase, b-glycan containing b-glycanase, cellulose containing cellulase, or lignin containing ligninase.

[0056] The enzyme-containing bio-derived particles of the present disclosure can be administered to a subject to treat a disease or disorder of the subject. Exemplary diseases or disorders include, but are not limited to, osteoarthritis (e.g., knee osteoarthritis, finger osteoarthritis); sports medicine diseases / disorders (e.g., frozen shoulder, tennis elbow (lateral epicondylitis), golfer's elbow (medial epicondylitis), pitcher's elbow (flexor tendinitis), Achilles tendinopathy, plantar fasciitis, symptomatic accessory navicular pain, hamstring tendinopathy, jumper's knee (patellar tendinitis), runner's knee (patellofemoral pain syndrome (PFPS)), pes anserine bursitis (knee pain), posterior tibial tendon disease, wrist (TFCC - triangular fibrocartilage complex) tendon disease, trigger finger (stenosing flexor tenosynovitis)); and orthopedic diseases / disorders (e.g., hemarthrosis). In another embodiment, the enzyme-containing bio-derived particles of the present disclosure are used in an embolization procedure for a subject in need thereof. Exemplary embolization procedures include, but are not limited to, embolization procedures in combination with TACE (wherein the particles of the present disclosure maximize the therapeutic effect by ensuring that the chemotherapeutic agent remains in place during absorption), prostate artery embolization, and uterine artery embolization.

[0057] In one embodiment, the enzyme-containing bio-derived particles of the present disclosure are administered to a subject to treat a tendinopathy of the subject. In another embodiment, the enzyme-containing bio-derived particles of the present disclosure are administered to a subject having a disease or disorder that would benefit from treatment with a temporary embolizing agent.

[0058] Alginate-based liquid embolization agents are considered promising alternatives to traditional embolization agents. Alginate in its pure form is highly biocompatible and its gelling properties can be controlled. They are naturally occurring polysaccharide copolymers composed of randomly 1-4 linked β-D-mannuronic acid (M-block)-α-L-guluronic acid (G-block) with various M:G ratios, which are typically found in various seaweeds. In the prior art disclosures, alginate is dissolved in the contrast agent iohexol (to confer radiopacity) and gelled into the form of a hydrocoil, which solidifies in the presence of a calcium chloride solution due to the ionic crosslinking of the carboxylate groups of the polysaccharide residues with Ca 2+ ions. All these components are mixed simultaneously at the treatment site to produce an in-situ gel mass. Subsequently, the gel can be dissolved using a mixture called EmboClear by the inventors, which is a mixture of alginate lyase and EDTA (ethylenediaminetetraacetic acid). The enzyme cleaves the polysaccharide chain at the glycosidic bond via a β-elimination mechanism, and EDTA decomplexes the ionic crosslinking by chelating Ca 2+ ions. The dissolving agent is administered at the embolization site and it completely clears the occluded blood vessel within a few minutes. The present invention addresses some aspects of the selective degradation of the embolization agent but causes some complications.

[0059] First, the procedure of using the Emboclear solution to degrade EmboGel introduces additional risks to the patient as they have to undergo an additional post-embolization procedure. Moreover, depending on the desired time interval between embolization formation and its dissolution, this may involve rescheduling the patient for a second visit and all the associated costs of reinserting the catheter procedure. Second, in some cases, such as in aneurysm therapy, the alginate gel may migrate to the parent artery during injection or after the post-embolization procedure, which may cause non-specific vascular occlusion (see, for example, Barnett et al., “A selectively dissolvable radiopaque hydrogel for embolic applications”; and U.S. Patent No. 9,220,761). Other bio-derived particles containing enzymes that can act on bio-derived particles will be expected to have similar complications.

[0060] Barnett et al. demonstrated that alginate-based embolization materials can be degraded in vivo by applying an alginate lyase-based composition. Purified alginate is dissolved in the contrast agent iohexol (to confer radiopacity) and gelled into the form of a hydrocoil, which solidifies in the presence of a calcium chloride solution due to the ionic crosslinking of the carboxylate groups of the polysaccharide residues with Ca 2+ ions. All these components are mixed simultaneously at the treatment site to produce an in-situ gel mass. Subsequently, the gel can be dissolved using EmboClear.

[0061] In U.S. Patent No. 9,220,761, the non-specific migration of the degraded / disintegrated alginate gel to other parts of the body occurs mainly due to the instantaneous / uncontrolled degradation / disintegration of EmboClear on EmboGel, causing the generation of particles of various sizes and being unable to be reabsorbed before they are distributed to distal positions away from the target, where EmboClear is ineffective due to dilution. If EmboGel is loaded with a bioactive agent / drug, an EmboClear dissolving agent needs to be administered separately to provide degradation-controlled release kinetics.

[0062] Boyan et al. reported methods and compositions of alginate particles composed of alginate lyase and stem cells (i.e., see PCT Publication No. WO 2012 / 071527 A2). Depending on the concentration of the incorporated enzyme, proteins secreted by stem cells or stem cells can be delivered into the body. Contrary to various embodiments of the present disclosure, Boyan's compositions cannot be lyophilized and sterilized without killing the stem cells therein. In Boyan's patent, Ca 2+ crosslinked alginate particles encapsulate alginate lyase and stem cells for the sustained release of proteins and stem cells. In this method, different amounts of alginate lyase are mixed with stem cells with alginates of different molecular weights at 1 - 4 °C for 1 minute and then gelled in a calcium chloride bath to obtain calcium-crosslinked alginate microspheres encapsulating stem cells that can self-degrade. To release the stem cells and the proteins they secrete, these particles are suspended in saline at 37 °C to activate the alginate matrix-degrading catalytic activity of alginate lyase. In addition, the alginate particles loaded with cells are treated with DMSO for cryopreservation in liquid nitrogen. This report provides in-depth understanding of the controlled degradation of alginate particles, but there are many drawbacks in using this method to produce scalable self-degradable alginate particles for embolization applications. It is observed that the reduction of temperature to 1 - 4 °C does not completely terminate the degradation activity of the enzyme. In addition, if the production of these particles needs to be scaled up proportionally, the incubation period of the enzyme with alginate will be longer. This reduces the viscosity of alginate, thus reducing the encapsulation of alginate lyase and also posing challenges to obtaining particles with a uniform shape. Similarly, the encapsulation of any proposed bioactive agents (such as anti-inflammatory agents and anti-cancer agents) will also be reduced. In addition, methods for encapsulating bioactive agents and post-processes after particle preparation, such as lyophilization and sterilization of degradable alginate particles, are not considered. To develop self-degrading particles, it is important to be able to store them in a dry and sterile form for a longer time, which can be reconstituted when in use and become activated after being introduced into the body. Therefore, there is a need for self-degrading particles that can degrade or exhibit a predictable rate of resorption without causing any non-specific occlusion in the body, act as a vehicle for releasing bioactive agents, and remain stable under desired storage conditions for a long time.

[0063] In addition, Kunjukunju et al. reported the use of ammonium sulfate for alginate lyase aggregates of various sizes (10 - 300 μm) and shapes (see, for example, Kunjukunju et al., "Cross-linked enzyme aggregates of alginate lyase: A systematic engineered approach to controlled degradation of alginate hydrogel." International Journal of Biological Macromolecules 115 (2018): 176 - 184). These aggregates were cross-linked using glutaraldehyde to produce insoluble, catalytically active alginate lyase aggregates. The resulting cross-linked aggregates were encapsulated in alginate hydrogel to effect their controlled degradation. However, the method described in that report may not be suitable for preparing the self-degrading particles of the present disclosure per se.

[0064] First, it is not possible to produce self-degrading particles of the desired size because the size and polydispersity of the aggregates of the enzyme cannot be encapsulated. Second, the method describes cross-linking enzyme aggregates with glutaraldehyde, which is a toxic agent that should be avoided in the preparation of compositions intended for human use. Third, the authors did not report any other methods to control the degradation of the bioderived particles, such as the molecular weight or viscosity of the biomaterial used to prepare the particles (e.g., sodium alginate for alginate bioderived particles), pretreatment of the enzyme with modifiers (metal ions) or other physicochemical parameters (e.g., pH and temperature), or improvement of the encapsulation efficiency of the enzyme. Finally, no work has been done to achieve the storage and shelf life of the resulting aggregates.

[0065] The present disclosure provides compositions and methods for preparing self-degradable cross-linked microspheres, wherein the microspheres are bioderived and loaded with an enzyme that acts on the microspheres. In one embodiment, the microspheres are further loaded with a bioactive agent and a cryoprotectant. This allows for the loading or encapsulation of the desired concentration of the enzyme and the bioactive agent to obtain customized degradation of the microspheres under physiological conditions. The present disclosure has many advantages compared to existing self-degrading particles and prior art alginate-based systems:

[0066] 1. Pretreating the enzyme under a combination of different conditions (pH, temperature, and metal ion inhibitors) that can reversibly inhibit the catalytic degradation activity allows for the controlled loading of the enzyme into the bioderived particles. This strategy provides a predictable and desired degradation rate of the bioderived particles, which is of utmost importance for applications that require the temporary use of self-degrading particles (e.g., embolization applications). For existing self-degrading particles and prior alginate-based systems, the use of a combination of different conditions to regulate the loading of the enzyme into the bioderived particles has not been described;

[0067] 2. Pretreatment of the enzyme reversibly inhibits the enzyme activity, thus stopping the exposure of the bio-derived particles to the active form of the enzyme for a desired length of time. This strategy can allow for the scaled-up production of these microspheres without prematurely degrading the bio-derived particles encapsulating the enzyme;

[0068] 3. The self-degrading property of the bio-derived microspheres containing the enzyme ensures that any by-products or particles can be reabsorbed and ultimately excreted through the kidneys. Thus, the risk of non-specific occlusion of blood vessels is minimized;

[0069] 4. The self-degradable bio-derived microspheres can also be loaded with anti-inflammatory agents, such as hyaluronic acid. Sustained release of the anti-inflammatory agent in the subject can relieve the pain of the subject. For example, sustained release of the anti-inflammatory agent at the embolization site can relieve neuropathic pain that may be caused by chronic inflammation.

[0070] 5. The composition of the self-degradable bio-derived microspheres also comprises a cryoprotectant. Inclusion of the cryoprotectant allows for lyophilization and subsequent sterilization without affecting the enzyme activity. These post-preparation processing steps of the microspheres produce a sterile composition that can be stored for a certain length of time and reconstituted at the time of use before administration into the body to reactivate the enzyme.

[0071] In addition to divalent metal ion crosslinking, photopolymerization methods can be used to prepare self-degradable bio-derived particle compositions having the same properties as those discussed above. This method can further improve the calibrated degradation of the bio-derived microspheres.

[0072] Definitions

[0073] As used herein, the terms "a", "an", or "the" are generally construed to cover both the singular and plural forms.

[0074] As used herein, the term "about" generally refers to a particular numerical value within an acceptable error range as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measuring system. For example, "about" can refer to a range of ±20%, ±10%, or ±5% of a given numerical value.

[0075] As used herein, the term "substantially" can refer to mainly or mostly, such as at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more.

[0076] As used herein, "carrier" or "vehicle" refers to a carrier material suitable for drug administration. Carriers and vehicles useful herein include any such materials known in the art, such as any liquid, gel, solvent, liquid diluent, solubilizer, surfactant, etc., which are non-toxic and do not interact with the other components of the composition in a harmful manner.

[0077] The term "therapeutically effective amount" generally can refer to the amount (or dose) of a compound or other therapy that, when administered to a subject in need thereof, is minimally sufficient to prevent, reduce, treat, or eliminate a condition or its risk. In some cases, the term "therapeutically effective amount" can refer to the amount of a compound or other therapy that is sufficient to have a preventive effect when administered to a subject. The therapeutically effective amount can vary; for example, it can vary depending on the condition of the subject, the weight and age of the subject, the severity of the disease condition, the mode of administration (e.g., subcutaneous delivery), etc., all of which can be determined by one of ordinary skill in the art.

[0078] As used herein, "treatment" includes: (i) preventing the occurrence of a pathological condition (e.g., prophylaxis); (ii) inhibiting a pathological condition or arresting its development; (iii) alleviating a pathological condition; and / or (iv) reducing the symptoms associated with a pathological condition.

[0079] The phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that, within the scope of reasonable medical judgment, are suitable for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0080] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Except for any conventional media or agents that are incompatible with the active ingredient, their use in the therapeutic compositions of the present disclosure is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0081] The term "pharmaceutically acceptable excipient" is intended to include vehicles and carriers capable of being co-administered with a compound to facilitate the performance of its intended function. The use of such media for pharmaceutically active substances is well known in the art. Examples of such vehicles and carriers include solutions, solvents, dispersion media, delaying agents, emulsions, etc. Any other conventional carrier suitable for use with a multi-binding compound also falls within the scope of the present disclosure.

[0082] Compositions and Methods

[0083] Crosslinked bio-derived microspheres

[0084] The present disclosure relates to loading enzymes into biomaterials, which can be gelled or crosslinked using any conventional methods used in the art, depending on the biomaterials used, to induce covalent, physical, ionic crosslinking, or a combination thereof, to form enzyme-loaded bio-derived microspheres. In one embodiment, a metal ion crosslinking agent (e.g., divalent metal ions) is used. In another embodiment, a homobifunctional crosslinking agent (e.g., disuccinimidyl suberate, succinimidyl tartrate, dithiobis(succinimidyl propionate), bismaleimidoethane, dithiobis(bismaleimidoethane), etc.) is used. In still another embodiment, a heterobifunctional crosslinking agent (e.g., MDS (m-maleimidobenzoyl-N-hydroxysuccinimide ester), GMBS (N-γ-maleimidobutyryloxy succinimide ester), EMCS (N-(ε-maleimidocaproyl)oxysuccinimide ester), sulfo-EMCS (N-(ε-maleimidocaproyl)oxysulfosuccinimide ester and its derivatives)) is used. In still another embodiment, crosslinking is formed by thermal gelation (e.g., via gelation of gelatin, agar, pectin, and other such biopolymers). In still another embodiment, crosslinking is formed by photoactivated photopolymerization or photocrosslinking (e.g., using aryl azides, bisaziridines, and their derivatives). Then, the crosslinked microspheres of the present disclosure can be rapidly dried and sterilized.

[0085] In one embodiment, a heterobifunctional crosslinking agent is used to crosslink bio-derived microspheres containing enzymes. In this embodiment, a biomaterial containing amine or carboxyl functional groups can be crosslinked using EDC-NHS click chemistry or any of the above heterobifunctional agents to form amide or other covalently crosslinked microspheres.

[0086] In another embodiment, thermal gelation is used to crosslink bio-derived microspheres containing enzymes. In this embodiment, the biomaterial is heated to melt it, the enzyme is added, the resulting mixture is emulsified in oil to form droplets, and the droplets are cooled to form microspheres. In one embodiment, an aqueous solution of agarose is used as the biomaterial, where the solution is heated to 40 °C to melt the agarose, the enzyme is added, the resulting mixture is emulsified in oil to form droplets, and the droplets are cooled to form microspheres.

[0087] To prepare divalent metal ion-crosslinked bio-derived microspheres loaded with enzymes that act on the microspheres, the enzyme is mixed with a biomaterial (precursor solution) that can be crosslinked by metal ions and dropped into a divalent metal ion gelling bath.

[0088] In the case of enzyme-loaded photocrosslinked bioderived microspheres, a biomaterial containing a photocrosslinkable moiety is mixed with an enzyme and a photoinitiator (precursor solution), which can be, for example, cast as droplets or injected using a microfluidic platform into different liquids (single emulsion or double emulsion) containing a surfactant or oil to form droplets. These droplets are irradiated with light of near-UV wavelengths (200 nm - 400 nm) for different times. After irradiation, the biomaterial containing the photocrosslinkable moiety crosslinks to form enzyme-loaded bioderived microspheres. In one embodiment, the photocrosslinkable moiety is an acrylate or methacrylate moiety.

[0089] In one embodiment, the microspheres of the present disclosure can also encapsulate an anti-inflammatory agent. In one embodiment, the anti-inflammatory agent is included during the preparation of the bioderived microspheres, and then the microspheres are crosslinked using the above crosslinking procedure. The degradation of these crosslinked enzyme-containing bioderived particles can be controlled by the composition of the precursor solution, the composition of the gelling bath, and the method of preparing these particles.

[0090] In one embodiment, the bioderived particles contain proteins or glycoproteins, such as albumin particles, collagen particles, gelatin particles, fibrinogen particles, or fibroin particles. In one embodiment, the bioderived particle-enzyme combinations are albumin containing peptidase, collagen or gelatin containing protease, gelatin containing matrix metalloproteinase, fibrinogen containing plasmin, or fibroin containing protease. In another embodiment, the bioderived particles contain polysaccharides, such as alginate particles, hyaluronic acid particles, pectin particles, starch particles, chitosan particles, agar / agarose particles, carrageenan particles, pullulan particles, dextran particles, β-glucan particles, cellulose particles, or lignin particles. In one embodiment, the bioderived particle-enzyme combinations are alginate containing alginate lyase (alginate lyase), hyaluronic acid containing hyaluronidase, pectin containing pectinase, starch containing amylase, chitosan containing chitosanase or lysozyme, agar / agarose containing agarase, carrageenan containing carrageenase, pullulan containing pullulanase, dextran containing dextranase, β-glucan containing β-glucanase, cellulose containing cellulase, or lignin containing ligninase.

[0091] In one embodiment, the bioderived particles are alginate microspheres loaded with alginate lyase. Thus, in one embodiment, the biomaterial is sodium alginate or methacrylate-alginate loaded with sodium alginate lyase. In one embodiment, sodium alginate is used as the biomaterial when the crosslinking agent is a divalent metal ion. In another embodiment, methacrylate-alginate is used as the biomaterial crosslinked by photoactivated photopolymerization.

[0092] Compositions and Methods for Preparing Enzyme - Loaded Divalent Metal Ion - Crosslinked Self - Degradable Biologically - Derived Microspheres

[0093] Composition of the precursor solution

[0094] The degradation of the enzyme-loaded crosslinked bioderived particles can be controlled by the composition of the precursor solution. In one embodiment, the concentration of the enzyme loaded into the bioderived microspheres can control the degradation of the crosslinked bioderived particles. In one embodiment, the enzyme can be pretreated with pH, temperature, metal ion inhibitors, organic and inorganic competitive inhibitors, non-inhibitors, end product inhibitors, or combinations thereof. In one embodiment, the pretreatment of the enzyme can control the degradation of the enzyme-loaded crosslinked bioderived particles.

[0095] In the precursor solution, the enzyme causes the decomposition of the biomaterial, which may affect the encapsulation of the enzyme within the bioderived particles. This also reduces the initial viscosity of the enzyme solution, which is important for both maintaining the encapsulation efficiency within the bioderived particles and obtaining particles of the desired size and shape. Thus, the enzyme can be pretreated at different pHs, low temperatures, and / or exposure to metal ion inhibitors prior to adding the precursor mixture to a divalent metal ion gelling bath for crosslinking.

[0096] Optimal catalytic activity of the alginate lyase or alginase used in the present disclosure is observed in the pH range of 6.8 - 7.5. To prevent the initial degradation of the biomaterial during the preparation of the enzyme-loaded bioderived particles, the pH of the enzyme-biomaterial solution can be reduced to 3.0. To carry out this process, a sodium acetate-acetic acid buffer with an ionic strength <1M, preferably <0.1M, and most preferably <0.01M is used, where the pH ranges from 3.7 - 5.6. Additionally, sodium hydroxide (>1M to <0.01M) or hydrochloric acid (>1M to <0.01M) can also be used to achieve the desired pH of the solution (pH 6.5 - 3.0). This results in a decrease or cessation of the enzyme catalytic activity. This regulation of catalytic activity can be attributed to the unfolding of the 3D conformation of the enzyme. By exposing the enzyme-loaded bioderived particles to an aqueous environment with a pH of 6.5 - 7.5, the terminated catalytic activity of the enzyme can be reversed / activated. The preferred buffer for reversing the activity of the enzyme is a phosphate buffer. At 20°C, the preferred ionic strength of the phosphate buffer is 0.01M, where the pH ranges from 6.5 - 7.5. Sodium hydroxide (>1M to <0.01M) or hydrochloric acid (>1M to <0.01M) can also be used to achieve the desired pH of the solution (pH 6.5 - 7.5). Additionally, brine or deionized water or an aqueous solution with a pH between 6.5 - 7.5 can also be used.

[0097] In combination with changing the pH of the solution, the temperature of the individual components of the precursor solution can be maintained at 1 - 4°C prior to mixing to inhibit the degradation of the biomaterial. After mixing the individual components, the temperature of the precursor solution can be maintained at 1 - 4°C to inhibit the degradation of the biomaterial. Note that the temperature will also affect the viscosity of the solution.

[0098] In addition to changing the pH and temperature of the precursor solution, the enzyme can be pretreated with metal ion inhibitors such as Cu 2+ , Zn 2+ and Fe 3 + . These metal ions can inhibit the enzyme activity (Inoue et al., “Functional identification of alginate lyase from the brown alga Saccharina japonica”, Sci. Rep. 2019; 9: 1-11).

[0099] Therefore, a combination of the above methods can be used to (reversibly or partially) inhibit the enzyme in the precursor solution. This can enhance the loading of the enzyme onto the bio-derived particles without degrading the particle matrix. The combination of these methods was not employed in the cited reports.

[0100] Other factors that improve the robustness of the biomaterial are the choice of divalent ions and the molecular weight / viscosity of the biomaterial. The molecular weight or viscosity of the biomaterial also affects the mechanical properties of the bio-derived particles (Farrés et al., “Formation kinetics and rheology of alginate fluid gels produced by in-situ calcium release,” Food Hydrocolloids 40 (2014): 76-84).

[0101] To achieve rapid degradation of the bio-derived particles with enzyme-loaded divalent metal ion complexation, a biomaterial with a low molecular weight / low viscosity can be used in the precursor solution. In one embodiment, using a biomaterial with a low molecular weight / low viscosity results in bio-derived particles that degrade within > 20 minutes to <= 4 hours. In one embodiment, using a biomaterial with a low molecular weight / low viscosity results in bio-derived particles that degrade within less than about 12 hours, less than about 10 hours, less than about 8 hours, less than about 6 hours, less than about 4 hours, less than about 3 hours, less than about 2 hours, or less than about 1 hour. To achieve a medium (5 days to 30 days) or slow (> 30 days) degradation period, a biomaterial with a high molecular weight / high viscosity can be used, which can be considered > 70 mPas.

[0102] The activity (units, U) of the treated (pH, temperature, and / or metal ion inhibitor exposed) enzyme mixed with biomaterials of different molecular weights in the precursor solution also regulates the degradation of the divalent metal ion crosslinked bio-derived microspheres. For rapid degradation (>20 minutes to <=4 hours) of the bio-derived microspheres, the preferred activity range of the enzyme can be 0.0075 U / mg to 0.25 U / mg of biomaterial. To obtain medium (5 days to 30 days) or slow (>30 days) degradation periods, the preferred ranges of enzyme activity are <0.005 U / mg to 0.025 U / mg of biomaterial and <0.0025 U / mg of biomaterial, respectively.

[0103] In embodiments where the biomaterial is alginate, the predetermined molecular weight of the alginate and the ratio (M / G) of the M (β-D-mannuronic acid) to G (α-L-guluronic acid) blocks can control the self-degradation of the resulting alginate microspheres. In particular, due to the geometry of the carboxylate residues, the G block has a higher affinity for divalent cations compared to the M block. The M and G contents of alginate vary widely and also vary in the sequence structure (G-block, M-block, and MG-block) (Ramos et al., “Effect of alginate molecular weight and M / G ratio in beads properties foreseeing the protection of probiotics”, Food Hydrocoll. 2018; 77:8-16). Generally, when compared to alginates with higher M / G ratios, alginates with higher G contents (lower M / G ratios) relative to the M content result in mechanically robust structures / capsules with low permeability and greater resistance to enzymatic degradation when crosslinked with cations.

[0104] In one embodiment, the enzyme-loaded bioderived particles are divalent metal ion-complexed alginate particles loaded with alginate lyase. To achieve rapid degradation (>20 minutes to <=4 hours) of these particles, a lower G content alginate with a low molecular weight / low viscosity (e.g., a higher M:G ratio) can be used in the precursor solution. In certain embodiments, the purified alginate contains more than 50% M content (β-D-mannuronic acid). The percentage of M content in the purified alginate can be 50%-80%, 55%-75%, and 60%-80%. To obtain a medium (5 days to 30 days) or slow (>30 days) degradation period, a higher G content alginate with a high molecular weight / viscosity (e.g., a lower M:G ratio) can be used. In certain embodiments, the purified alginate contains more than 50% G content (α-L-guluronic acid). The percentage of G content in the purified alginate can be 50%-80%, 55%-75%, and 60%-80%. A preferred M content can be in the range of about 55% to about 65% to obtain particle degradation over short and medium time periods. Without wishing to be bound by theory, it is believed that G residues bind metal ions more tightly than M residues. Thus, a higher M content will result in microspheres that may be less tightly crosslinked compared to corresponding microspheres with a higher G content and will therefore degrade faster as it is easier to displace the crosslinking ions and open the structure.

[0105] In embodiments where the biomaterial is alginate, the average molecular weight of the alginate polymer can be >200 kDa, preferably >100 kDa, and most preferably >30 kDa. At 20 °C, the viscosity of a 1% alginate solution can have a range of >25 mPa·s, preferably <1000 mPa·s, to prepare rapidly and slowly degrading divalent metal ion-complexed alginate particles loaded with alginate lyase.

[0106] In embodiments where the self-degrading bioderived particles are alginate particles loaded with alginate lyase, the activity (units, U) of the treated (pH, temperature, and metal ion inhibitor exposure) alginate lyase mixed with alginates of different molecular weights and M / G ratios (M (β-D-mannuronic acid) to G (α-L-guluronic acid) block (M / G) ratio) in the precursor solution also regulates the degradation of divalent metal ion-crosslinked alginate microspheres. The activity of the alginate lyase can range from 0.025 U / mg to 1 U / mg alginate. For rapid degradation (>20 minutes to <=4 hours) of the alginate microspheres, the preferred activity of the enzyme can range from 0.0075 U / mg to 0.25 U / mg alginate. To obtain medium (5 days to 30 days) or slow (>30 days) degradation periods, the preferred ranges of enzyme activity can be 0.005 U / mg to 0.0025 U / mg alginate and <0.0025 U / mg to 0.005 U / mg alginate, respectively. Although not wishing to be bound by theory, it is believed that the higher the activity of the enzyme (the more encapsulated), the faster the enzyme degrades the microspheres.

[0107] Use of a bioactive agent encapsulated within a bioderived particle

[0108] In one embodiment, the self-degrading enzyme-loaded bioderived particles of the present disclosure can encapsulate a bioactive agent, such as an anti-inflammatory agent (including NSAIDs and non-NSAIDs), a chemotherapeutic agent, a corticosteroid, or an antioxidant, which acts to provide local pain relief when administered to a subject. Exemplary NSAID anti-inflammatory agents include, but are not limited to, ibuprofen, flurbiprofen, meloxicam, nabumetone, etodolac, ketorolac, ketoprofen, diflusinal, naproxen, diclofenac, celecoxib, mefenamic acid, etoricoxib, indomethacin, and aspirin. Exemplary non-NSAID agents include, but are not limited to, arnica, curcurmin, bromelain, and acetaminophen. Exemplary corticosteroids include, but are not limited to, methylprednisolone, dexamethasone, triamcinolone, betamethasone, beclomethasone, and hydrocortisone. Exemplary antioxidants include, but are not limited to, glutathione, α-tocopherol, ergothioneine, N-acetylcysteine, ascorbic acid, vitamin A, vitamin E, allopurinol, melatonin, resveratrol, bucillamine, turmeric, lycopene, lipoic acid, cobalamin, flavonoids, quercetin, ebselen, and edaravone.

[0109] Previous reports have shown that high molecular weight hyaluronic acid (100 - 500 kDa) or degradation products of hyaluronic acid exhibit anti-inflammatory and immunosuppressive activities. Thus, when administered to a subject, hyaluronic acid encapsulated within the enzyme-loaded bioderived particles of the present invention can provide pain relief. Similarly, other anti-inflammatory drugs or antioxidants are also expected to provide pain relief when administered to a subject.

[0110] A bioactive agent, such as high molecular weight hyaluronic acid, can be added to the aforementioned precursor solution. This involves adding the bioactive agent at a concentration of 1% wt to 20% wt of the biomaterial concentration in the precursor solution. The bioactive agent can be encapsulated upon crosslinking in a divalent metal ion gelling bath. In one embodiment, high molecular weight hyaluronic acid is encapsulated within the enzyme-loaded divalent metal ion crosslinked bioderived microspheres described elsewhere herein.

[0111] In one embodiment, the bioactive agent provides local pain relief in a subject suffering from tendinopathy. In another embodiment, the bioactive agent provides local pain relief in a subject during an embolization medical intervention. Due to the degradation of the enzyme-loaded bioderived microspheres, the encapsulated bioactive agent can be released at the embolization site or at the site of tendinopathy or osteoarthritis. For example, many embolization medical interventions cause neuropathic pain, which can be alleviated by using hyaluronic acid or its degradation products, alginates and their degradation products, NSAIDs, non-NSAIDs, corticosteroids, and antioxidants. These pharmacologically active molecules can relieve pain caused by tendinopathy and osteoarthritis.

[0112] In another embodiment, the self-degrading enzyme-loaded bioderived particles of the present disclosure are formed from a polysaccharide biomaterial, resulting in polysaccharide particles encapsulating an enzyme that can act on the polysaccharide, wherein the particles do not encapsulate an anti-inflammatory agent or an antioxidant. In one embodiment, the enzyme degrades the polysaccharide particles to form oligosaccharides with anti-inflammatory effects. Thus, in some embodiments, even though the particles of the present disclosure do not encapsulate an anti-inflammatory agent or an antioxidant, when administered to a subject, the particles of the present disclosure are capable of providing an anti-inflammatory effect and / or pain relief.

[0113] Composition of the gelling bath

[0114] As described elsewhere herein, under appropriate conditions (low temperature, pH, or metal ions), a solution of a precursor enzyme - biomaterial (for alginate biomaterials and alginate lyase, appropriate molecular weight and optional M / G ratio) needs to be gelled in a divalent metal ion bath to form the microspheres of the present disclosure. In some embodiments, a bioactive agent is added to the precursor enzyme - biomaterial solution, and the resulting mixture is gelled in a divalent metal ion bath to form microspheres. The obtained particle size can be >40 μm, <200 μm, but <2000 μm. To reduce the degradation of the divalent cross - linked bio - derived microspheres, the temperature and pH of the gelling bath can be maintained at 1 - 4 °C and 3.5 to 4.0, respectively. A buffer with the above - mentioned ionic strength can be used to maintain the pH. The composition and conditions of the gelling bath are important for preparing the desired self - degrading bio - derived particles. The divalent metal ion component of the gelling bath composition can be selected from Cu 2+ , Ba 2+ , Sr 2+ , Ca 2+ , Co 2+ , Ni 2+ , Mn 2+ and Mg 2+ (Lee et al., “Alginate: properties and biomedical applications,” Progress in polymer science 37, no.1 (2012): 106 - 126; and Brus et al., “Structure and dynamics of alginate gels cross - linked by polyvalent ions probed via solid state NMR spectroscopy”, Biomacromolecules 18, no.8 (2017): 2478 - 2488). The choice of divalent cation can also affect the cross - linking of the biomaterial matrix outside the bio - derived particles. In one embodiment, the bio - derived particles comprise alginate particles loaded with alginate lyase, wherein the binding strength of the divalent metal ion to alginate is given in decreasing order of Cu 2+ >Ba 2+ >Sr 2+ >Ca 2+ >Co 2+ >Ni 2+ >Mn 2+ >Mg 2+ . Preferred metal cations are Ba 2+ and Ca 2+。These metal ions can be used at different concentrations ranging from 0.1% w / v to 10% w / v. The preferred concentration of divalent metal ions can be from 0.001% w / v to 2% w / v. In another embodiment, the bio-derived particles comprise pectin loaded with an enzyme (such as pectinase) that acts on pectin, wherein the binding strength of the divalent metal ion to the pectin depends on the pectin source. In one embodiment, the pectin is nopal pectin, wherein the divalent metal ion binding strength is given in decreasing order of Ca 2+ >Cu 2+ >Zn 2+ >Cr 3+ >Ni 2+ >Pb 2+ >Cd 2+ In one embodiment, the pectin is from citrus and sugar beet sources, wherein the divalent metal ion binding strength is given in decreasing order of Cu 2+ ≈Pb 2+ >>Zn 2+ >Cd 2+ ≈Ni 2+ ≥Ca 2+ These metal ions can be used at different concentrations ranging from 0.1% w / v to 10% w / v.

[0115] Water content and sterilization of bio-derived particles

[0116] The shelf life of enzyme-loaded self-degrading bio-derived microspheres can be improved by dehydrating the microspheres to make them substantially water-free and sterilizing the dehydrated microspheres.

[0117] The microspheres can be dehydrated using any technique known to those skilled in the art. In one embodiment, the microspheres are lyophilized. In another embodiment, the microspheres are dehydrated or dried using supercritical CO2. In this method, the crosslinked microspheres are treated with ethanol, acetone, or an ethanol:water mixture in the range of 10:90 to 98:2 to displace and replace water. The treated crosslinked particles are further processed by supercritical CO2. This method involves using pressurized liquid CO2 to remove the solvent in a stepwise manner. Here, the particles are placed in a stainless steel pressure vessel, exposed to liquid CO2, and heated at a specific temperature (preferably about 35°C to about 45°C) for 3 to 4 hours, and then the chamber is depressurized to remove the solvent. A cryoprotectant (such as trehalose) can be used only for preserving the enzyme.

[0118] In one embodiment, the microspheres are dried such that the water content of each microsphere is less than about 15%, less than about 12%, less than about 10%, less than about 8%, less than about 6%, less than about 4% or less than about 1% by mass. In one embodiment, the water content of each microsphere is between 1% and 10% by mass. Without wishing to be bound by theory, it is believed that if the water content of the microspheres is too high, the enzyme structure will denature upon sterilization, and if the water content is too low, the microspheres will not hydrate properly and the enzyme activity will be affected (a small amount of water is required to maintain the 3D conformation and activity of the enzyme).

[0119] For these post-preparation processes, cryoprotectants can be added to the precursor solution (described elsewhere herein) and the gelling bath. The addition of cryoprotectants is important in many ways. First, it helps to maintain the sphericity and mechanical robustness of the enzyme-loaded bioderived particles during the dehydration process. Second, it maintains the 3D conformation of the enzyme and thus the enzyme activity at the very low temperatures and freeze-thaw cycles used during lyophilization. The restoration of the microsphere shape after lyophilization is considered to be a problem. This is not unexpected as high water content gels shrink during lyophilization and generally cannot re-establish their original shape upon rehydration. The addition of cryoprotectants (such as sugars and polymers) can compensate for this to help maintain the porous structure during the sublimation of the internal water within the structure. In addition, it has been observed that when the enzyme is lyophilized without the addition of cryoprotectants / cryopreservation media, the residual activity of the enzyme is significantly reduced. Thus, the use of cryoprotectants also helps with the rapid restoration of shape upon reconstitution in an aqueous medium and enables the retention of the functionality of the active ingredients including the enzyme.

[0120] The microspheres can be sterilized using any technique known to those skilled in the art. In one embodiment, the microspheres are sterilized using irradiation. In one embodiment, the irradiation is electron beam irradiation. In another embodiment, the irradiation is γ-irradiation. In one embodiment, the γ-irradiation is between about 15 kGy and about 25 kGy. In another embodiment, the γ-irradiation is below 15 kGy, preferably equal to or below 6 kGy, or between about 6 kGy and about 10 kGy.

[0121] Composition of the precursor and gelling solutions containing cryoprotectants

[0122] The untreated or pretreated precursor enzyme-biomaterials and gelling solutions can be mixed with cryoprotectants (such as hydroxypropyl-β-cyclodextrin, trehalose, polyvinylpyrrolidone (PVP), and dextran) in different ratios. In one embodiment, the average molecular weight range of polyvinylpyrrolidone is >1 kDa to <40 kDa, preferably >1 kDa to <25 kDa, and more preferably the molecular weight is about 8 kDa. In one embodiment, the average molecular weight of dextran is 70 kDa.

[0123] Table 1-3 describes the composition of cryoprotectants in the precursor solution (Table 1), the gelling solution (Table 2), and the lyophilization solution (Table 3). The preferred concentrations of cryoprotectants in the precursor solution and the gelling bath are described as % w / v of the biomaterial concentration, which can be from about 0.1% w / v to about 4% w / v, and for the divalent metal ion concentration is from about 0.1% w / v to about 10% w / v. The concentration ranges of trehalose, hydroxypropyl-β-cyclodextrin, PVP, and dextran can be from about 0.1% w / v to about 20% w / v, from about 0.1% w / v to about 2% w / v, from about 0.1% w / v to about 1% w / v, and from about 0.1% w / v to about 1% w / v, respectively. Other cryoprotectants (such as glucose, lactose, maltodextrin, mannitol, ethylene glycol, and polyethylene glycol) can be used as stabilizer 1 in similar proportions as those discussed above. In one embodiment, glucose, lactose, maltodextrin, mannitol, ethylene glycol, or polyethylene glycol stabilizer 1 is used together with stabilizer 2.

[0124] Table 1. Composition of cryoprotectants in the precursor solution

[0125]

[0126] Table 2. Composition of cryoprotectants in the gelling solution

[0127]

[0128] Table 3. Composition of cryoprotectants in the lyophilization solution

[0129]

[0130] These cryoprotectant components can be mixed with the untreated and pretreated precursor solutions for 15 minutes to 1 hour, and then added to the gelling bath containing cryoprotectants for crosslinking with divalent metal ions to form enzyme-loaded and cryoprotectant-containing self-degrading bioderived microspheres. These microspheres can be dehydrated to obtain dry particles with a moisture content of about 10% mass or less, about 8% mass or less, about 6% mass or less, about 4% mass or less, about 3% mass or less, preferably about 2% mass or less, and more preferably about 1% mass or less. Under dry conditions sealed in vials, the dry particles can be further sterilized, such as by γ or electron beam radiation as described elsewhere herein. The dehydrated and sterilized enzyme-loaded divalent metal ion-crosslinked bioderived microspheres can be stored for ≤24 months.

[0131] Compositions and Methods for Preparing Enzyme - Loaded Photo - Crosslinked Self - Degradable Biologically - Derived Microspheres Composition of the precursor solution

[0132] Enzyme-loaded self-degrading photocrosslinked bioderived particles can be prepared using a biomaterial comprising a photocrosslinkable moiety and a photoinitiator. In one embodiment, the photocrosslinkable moiety is an acrylate or methacrylate moiety. Degradation of the photocrosslinked particles can be regulated by controlling the composition of the precursor solution, which comprises (i) an enzyme loaded into the photocrosslinked bioderived microspheres (which can be pretreated with pH, temperature, metal ion inhibitors, competitive inhibitors, noncompetitive inhibitors, and end-product inhibitors) and (ii) a biomaterial of a predetermined molecular weight comprising the photocrosslinkable moiety. In one embodiment, degradation of the photocrosslinked particles can also be regulated by varying the ratio of photoinitiator to biomaterial concentration and by varying the duration of light irradiation that affects particle crosslinking. In embodiments where the biomaterial comprising the photocrosslinkable moiety is alginate, the ratio of M (β-D-mannuronic acid) and G (α-L-guluronic acid) blocks (M / G) can also be used to regulate degradation of the resulting photocrosslinked particles.

[0133] Similar to bioderived microspheres crosslinked with divalent metal ions, the enzyme-containing precursor solution may degrade the biomaterial. This may reduce the viscosity of the biomaterial, decrease the enzyme loading capacity of the biomaterial, and adversely affect the size and shape of the particles. Specifically, in the precursor solution, the enzyme causes decomposition of the biomaterial, which can affect enzyme encapsulation into the bioderived particles. This also reduces the initial viscosity of the biomaterial solution, which is important for maintaining the encapsulation efficiency within the bioderived particles as well as for obtaining particles of a desired size and shape. Thus, the precursor solution can be pretreated at different pHs, low temperatures, and / or by exposure to metal ion inhibitors, competitive inhibitors, noncompetitive inhibitors, or end-product inhibitors before being subjected to light irradiation, or it can be left untreated. In one embodiment, the biomaterial is alginate, and alginate lyase in the precursor solution degrades the alginate.

[0134] All pretreatment procedures for the precursor solution are the same as those mentioned for the preparation of divalent metal ion-crosslinked microspheres described elsewhere herein.

[0135] Optimal catalytic activity of the alginate lyase was observed in the pH range of 6.8 - 7.5. To prevent the initial degradation of the biomaterial during the preparation of the enzyme-loaded bioderived particles, the pH of the enzyme-biomaterial solution can be reduced to 3.0. For this process, a sodium acetate - acetic acid buffer with an ionic strength < 1M, preferably < 0.1M, and most preferably < 0.01M is used, where the pH range is 3.7 - 5.6. Additionally, sodium hydroxide (> 1M to < 0.01M) or hydrochloric acid (> 1M to < 0.01M) can also be used to achieve the desired pH (pH 6.5 - 3.0) of the solution. This results in a decrease or cessation of the enzyme catalytic activity. This regulation of catalytic activity can be attributed to the unfolding of the 3D conformation of the enzyme. By exposing the enzyme-loaded bioderived particles to an aqueous environment with a pH of 6.5 - 7.5, the terminated catalytic activity of the enzyme can be reversed / activated. The preferred buffer for reversing the enzyme activity is a phosphate buffer. At 20 °C, the preferred ionic strength of the phosphate buffer is 0.01M, where the pH range is 6.5 - 7.5. Sodium hydroxide (> 1M to < 0.01M) or hydrochloric acid (> 1M to < 0.01M) can also be used to achieve the desired pH (pH 6.5 - 7.5) of the solution. Additionally, brine or deionized water or an aqueous solution with a pH between 6.5 and 7.5 can also be used.

[0136] In combination with changing the pH of the solution, before mixing, the temperature of the individual components of the precursor solution can be maintained at 1 - 4 °C to inhibit the degradation of the biomaterial. After mixing the individual components, the temperature of the precursor solution can be maintained at 1 - 4 °C to inhibit the degradation of the biomaterial. Note that the temperature will also affect the viscosity of the solution.

[0137] In addition to changing the pH and temperature of the precursor solution, the enzyme can be pretreated with metal ion inhibitors (such as Cu 2+ , Zn 2+ and Fe 3 + ). These metal ions can inhibit the activity of the enzyme (Inoue et al., “Functional identification of alginate lyase from the brown alga Saccharina japonica”, Sci. Rep. 2019; 9:1 - 11).

[0138] In combination with varying pH and temperature, other types of organic and inorganic inhibitors, including competitive, non - competitive, and end - product inhibitors, can also be added at different stages of particle synthesis (e.g., in the precursor solution, gelling solution, or lyophilized solution).

[0139] Synthesis of self - degrading photocrosslinked bioderived microspheres

[0140] To prepare self - degrading, photocrosslinkable, bio - derived microspheres, water - soluble photoinitiators such as Irgacure 2959, Irgacure 184, Irgacure 651, Irgacure 369, and Irgacure 907 can be used. The preferred photoinitiator is Irgacure 2959. These photoinitiators are activated under ultraviolet light irradiation with wavelengths ranging from 320 nm to 410 nm to form free radicals, but the ideal wavelength is 365 nm. The degradation of the microspheres also depends on the concentration of the biomaterial containing photocrosslinkable groups, the concentration of the photoinitiator, and the duration of UV irradiation. The concentrations of the biomaterial (such as alginate) containing photocrosslinkable groups and the photoinitiator in the precursor solution can be in the ranges of 0.1% w / v to 4% w / v and 0.1% w / v to 1.5% w / v, respectively. For rapidly degrading enzyme - loaded bio - derived microspheres (>20 minutes to <4 hours), the preferred concentrations of the biomaterial and the photoinitiator can be 1% w / v to 1.5% w / v and 0.1% w / v to 0.3% w / v, respectively. For microspheres with a medium degradation period (5 days to 30 days), the preferred concentrations of the biomaterial and the photoinitiator can be 2% w / v to 3% w / v and 0.4% w / v to 0.8% w / v, respectively. For slowly degrading enzyme - loaded bio - derived particles, the preferred concentrations of the biomaterial and the photoinitiator can be 3% w / v to 4% w / v and 0.9% w / v to 1.5% w / v, respectively. In addition, the duration of UV irradiation can be in the range of >10 seconds to <10 minutes, where the preferred duration for rapidly degrading microspheres can be <1 minute, 1 - 5 minutes for medium - degrading microspheres, and >5 minutes but <10 minutes for slowly degrading microspheres.

[0141] Combined with the concentration of the photoinitiator and the duration of UV irradiation, the degradation of the alginate-alginate lyase microspheres also depends on the G / M ratio and viscosity of the purified alginate. The percentage of the content of M (β-D-mannuronic acid) in the purified alginate can be 50%-80%, 55%-75% or 60%-80%. The preferred M content can be in the range of 55% to 65% to obtain particle degradation within shorter and medium time periods. To obtain a medium (5 days to 30 days) or slow (>30 days) degradation period, alginates with a higher G content (e.g., a lower M:G ratio) having a high molecular weight / viscosity can be used. In certain embodiments, the purified alginate contains more than 50% G content (α-L-guluronic acid). The percentage of the G content in the purified alginate can be 50%-80%, 55%-75% and 60%-80%. The alginate microspheres with rapid degradation (>20 minutes to <4 hours) have a low molecular weight / low viscosity (<70 mPas to >5 mPas). To achieve a medium (5 days to 30 days) or slow (>30 days) degradation period, biomaterials with a high molecular weight / high viscosity, which can be considered >70 mPas, can be used.

[0142] The method for preparing the microspheres can include a droplet casting technique, where droplets of the precursor solution can be printed on a superhydrophobic surface (e.g., PTFE), or generated using, for example, a single emulsion or double emulsion - microfluidic platform. After generating droplets of the desired size, UV light can be used to crosslink the biomaterial to form spherical enzyme-loaded bioderived microspheres.

[0143] In one embodiment, the method for preparing the microspheres can include an electroencapsulation process. In one embodiment, the electroencapsulation process includes passing the precursor solution through a needle with an electrostatic potential to form droplets. In one embodiment, the precursor solution contains the enzyme and biomaterial described elsewhere herein. After generating droplets of the desired size, divalent metal ions can be used to crosslink the biomaterial to form enzyme-loaded bioderived microspheres. In some embodiments, the electrostatic potential is between about 0.1 kV and about 20 kV, about 0.1 kV and about 18 kV, about 0.1 kV and about 16 kV, about 0.1 kV and about 14 kV, about 0.1 kV and about 12 kV, about 0.1 kV and about 10 kV, about 0.1 kV and about 8 kV, about 0.1 kV and about 6 kV, about 1 kV and about 6 kV, about 1 kV and about 4 kV, about 2 kV and about 4 kV or about 3 kV.

[0144] Use of a bioactive agent encapsulated within a bioderived particle

[0145] In one embodiment, the enzyme-loaded self-degrading bioderived particles of the present disclosure can encapsulate an anti-inflammatory agent or an antioxidant, which functions to provide local pain relief when administered to a subject. In one embodiment, the enzyme-loaded self-degrading bioderived particles of the present disclosure can encapsulate a chemotherapeutic agent. Previous reports have shown that high molecular weight hyaluronic acid (100 - 500 kDa) or its degradation products exhibit anti-inflammatory and immunosuppressive activities. Thus, when administered to a subject, the use of hyaluronic acid encapsulated within the enzyme-loaded bioderived particles of the present invention can provide pain relief. Similarly, other bioactive agents, including anti-inflammatory agents, NSAIDs, non-NSAIDs, corticosteroids, and antioxidants, are also expected to provide pain relief when administered to a subject. Exemplary anti-inflammatory agents, NSAIDs, non-NSAIDs, corticosteroids, and antioxidants are described elsewhere herein.

[0146] A bioactive agent, such as high molecular weight hyaluronic acid, can be added to the aforementioned precursor solution. This involves adding the bioactive agent to the precursor solution at a concentration of 1% wt to 20% wt of the biomaterial concentration. The bioactive agent can be encapsulated upon crosslinking in a divalent metal ion gelling bath. In one embodiment, high molecular weight hyaluronic acid is encapsulated within the enzyme-loaded divalent metal ion crosslinked bioderived microspheres described elsewhere herein.

[0147] In one embodiment, the bioactive agent provides local pain relief in a subject suffering from tendinopathy. In another embodiment, the bioactive agent provides local pain relief in a subject during an embolization medical intervention. Due to the degradation of the enzyme-loaded bioderived microspheres, the encapsulated bioactive agent can be released at the embolization site or at the tendinopathy or osteoarthritis site. For example, many embolization medical interventions cause neuropathic pain, which can be alleviated by using hyaluronic acid or its degradation products, alginates and their degradation products, NSAIDs, non-NSAIDs, corticosteroids, or antioxidants. These pharmacologically active molecules can relieve pain caused by tendinopathy and osteoarthritis.

[0148] In another embodiment, the self-degrading bioderived particles of the present disclosure comprise carbohydrate particles that encapsulate an enzyme that acts on the carbohydrate, wherein the particles do not encapsulate an anti-inflammatory agent or an antioxidant. In one embodiment, the enzyme degrades the carbohydrate particles to form oligosaccharides with anti-inflammatory effects. Thus, in some embodiments, even though the particles of the present disclosure do not encapsulate an anti-inflammatory agent or an antioxidant, when administered to a subject, the particles of the present disclosure are capable of providing an anti-inflammatory effect and / or pain relief.

[0149] Methods for manufacturing bioderived particles and enzyme-encapsulated bioderived particles using microfluidics

[0150] Enzyme-loaded bioderived particles described herein can be produced by droplet microfluidics, which provides precise control over the shape, size, and morphology of the resulting biocaterial droplets. Generally, a biocaterial solution is mixed with water-soluble Ca-EDTA or water-insoluble CaCO3 particles and emulsified in an oil phase on a microfluidic platform to produce droplets of the biocaterial solution with a desired size and shape. The droplets of the biocaterial can be crosslinked to divalent Ca released from Ca-EDTA or CaCO3 under acidic conditions. 2+ ions. This crosslinking can be carried out by "on-chip" or "off-chip" methods to produce Ca 2+ -crosslinked bioderived particles.

[0151] However, since enzymes can be effectively encapsulated in bioderived particles in an acidic pH environment, this conventional method is difficult to use. Thus, if enzymes are encapsulated in bioderived particles using the above conventional method, the enzyme-biocaterial mixture containing Ca-EDTA or CaCO3 will become gelled under acidic conditions and the solution cannot pass through the microfluidic platform to produce droplets of the biocaterial precursor solution. To overcome this drawback, a new method is proposed below to successfully produce enzyme-encapsulated bioderived particles using microfluidics.

[0152] The encapsulation of enzymes into the bioderived particles disclosed herein is carried out using the droplet microfluidics method, wherein a precursor solution is prepared in a buffer at pH 10 at a temperature of 1-4 °C, and wherein the precursor solution contains a biocaterial, an enzyme, and Ca-EDTA or CaCO3. In one embodiment, the precursor solution contains an excipient. In another embodiment, an excipient is used in the solution to wash the enzyme-encapsulated bioderived particles formed from the precursor solution. In one embodiment, the enzyme-encapsulated bioderived particles formed from the precursor solution are washed with water containing an excipient. The biocaterial can have a predetermined molecular weight. In an embodiment where the biocaterial is alginate, the alginate can have a predetermined G / M ratio. The concentration of Ca-EDTA or CaCO3 ranges from 1 M to 0.01 M, and a preferred concentration is from 0.05 M to 0.1 M.

[0153] The activity of alginate lyase can range from 0.025 U / mg to 1 U / mg alginate. For rapid degradation of alginate microspheres >20 minutes to <4 hours, the preferred activity of the enzyme can range from 0.075 mU / mg to 0.25 U / mg alginate. To obtain a medium (5 days to 30 days) or slow (>30 days) degradation period, the preferred ranges of enzyme activity can be <0.005 U / mg to ≥0.0025 U / mg alginate and <0.0025 U / mg alginate, respectively.

[0154] The pH range of the buffer solution is 8.0 - 13.0, and the preferred range of the buffer is 9.0 - 11.0. The buffer solution can be any buffer solution known to those skilled in the art. Common buffers that can be used to prepare biomaterials, enzymes, and Ca-EDTA / CaCO3 solutions are disodium hydrogen phthalate / sodium dihydrogen orthophosphate, sodium barbital / hydrochloric acid, dipotassium hydrogen phthalate / sodium dihydrogen orthophosphate, potassium dihydrogen orthophosphate / sodium hydroxide, sodium barbital / hydrochloric acid, tris(hydroxymethyl)aminomethane / hydrochloric acid, sodium tetraborate / hydrochloric acid, glycine / sodium hydroxide, sodium carbonate / sodium bicarbonate, sodium tetraborate / sodium hydroxide, sodium bicarbonate / sodium hydroxide, disodium hydrogen orthophosphate / sodium hydroxide, and potassium chloride / sodium hydroxide. The most preferred buffer system is sodium bicarbonate / sodium hydroxide. The ionic strength of the buffer is <1M, and the preferred range is <0.5M to >0.05M, and most preferably ≤0.1M. The preferred temperature range is <10°C, and most preferably >1°C to <4°C.

[0155] The above conditions allow for the generation of biomaterial droplets and overcome the challenges encountered in conventional methods for preparing bio-derived particles encapsulating enzymes in two ways: (a) these conditions inhibit the activity of the enzyme and thus prevent the initial degradation of the biomaterial in the precursor solution, and (b) these conditions prevent the release of Ca 2+ ions from Ca-EDTA or CaCO3. Additionally, the precursor solution is passed through a suitable microfluidic chip with oil to form biomaterial droplets (water-in-oil emulsion method). These droplets are crosslinked with divalent Ca 2+ ions by exposure to acetic acid at a concentration of 0.01% v / v to 5% v / v. The preferred range of acetic acid concentration is 1% to 2% v / v acetic acid. Upon exposure to acetic acid, Ca 2+ ions are released from Ca-EDTA or CaCO3 and bind to the egg box of the biomaterial to form Ca 2+ -crosslinked enzyme-loaded bio-derived particles. The particles are then washed with deionized water containing excipients to remove the acid. If desired, the particles are further crosslinked in a calcium chloride solution with a concentration in the range of 2% w / v to 10% w / v. The washed particles are suspended in a solution containing excipients for a duration of 6 - 24 hours and dried. The dried particles can be reconstituted in a neutral pH buffer to activate the enzyme and initiate the degradation of the enzyme-loaded bio-derived particles.

[0156] Water content and sterilization of bio-derived particles

[0157] The shelf life of enzyme-loaded self-degrading enzyme-loaded bio-derived microspheres can be improved by dehydrating the microspheres to make them substantially water-free and sterilizing the dehydrated microspheres.

[0158] The microspheres can be dehydrated using any technique known to those skilled in the art. In one embodiment, the microspheres are lyophilized. In another embodiment, the microspheres are dehydrated using supercritical CO2. In one embodiment, the microspheres are dried such that the water content of each microsphere is less than about 15%, less than about 12%, less than about 10%, less than about 8%, less than about 6%, less than about 4% or less than about 1% by mass. In one embodiment, the water content of each microsphere is between 1% and 10% by mass. Without wishing to be bound by theory, it is believed that if the water content of the microspheres is too high, the enzyme will denature upon sterilization, and if the water content is too low, the microspheres will not hydrate properly and the enzyme activity will also be affected (a small amount of water is required to maintain the 3D conformation and activity of the enzyme).

[0159] For these post-preparation processes, cryoprotectants can be added to the precursor solution (described elsewhere herein). The addition of cryoprotectants is important in many ways. First, it helps to maintain the sphericity and mechanical robustness of the enzyme-loaded bioderived particles during the dehydration process. Second, it maintains the 3D conformation of the enzyme and thus the enzyme activity at the extremely low temperatures and freeze-thaw cycles used during lyophilization. The restoration of the microsphere shape after lyophilization is considered a problem. This is not unexpected since high water content gels shrink during lyophilization and generally cannot re-establish their original shape upon rehydration. The addition of cryoprotectants (such as sugars and polymers) can compensate for this to help maintain the porous structure during the sublimation of internal water within the structure. In addition, it has been observed that when the enzyme is lyophilized without the addition of cryoprotectants / cryopreservation media, the residual activity of the enzyme is significantly reduced. Thus, the use of cryoprotectants also helps with the rapid restoration of shape upon reconstitution in an aqueous medium and enables the retention of the functionality of the active ingredients including the enzyme.

[0160] The microspheres can be sterilized using any technique known to those skilled in the art. In one embodiment, the microspheres are sterilized using irradiation. In one embodiment, the irradiation is electron beam irradiation. In another embodiment, the irradiation is γ-irradiation. In one embodiment, the γ-irradiation is between about 15 kGy and about 25 kGy. In another embodiment, the γ-irradiation is less than 15 kGy, preferably equal to or less than 6 kGy.

[0161] Composition of the precursor solution containing cryoprotectants

[0162] An untreated or pre-treated proenzyme-biomaterial solution (as described elsewhere herein) can be mixed with cryoprotectants (such as hydroxypropyl-β-cyclodextrin, trehalose, polyvinylpyrrolidone, and dextran) in different ratios. In one embodiment, the molecular weight of polyvinylpyrrolidone is 40 kDa (PVP 40 kDa). In one embodiment, the average molecular weight range of polyvinylpyrrolidone is >1 kDa to <40 kDa, preferably >1 kDa to <25 kDa, and more preferably the molecular weight is about 8 kDa. In one embodiment, the average molecular weight of dextran is 70 kDa.

[0163] Tables 1-3 describe the composition of cryoprotectants in the precursor solution (Table 1), the gelling solution (Table 2), and the lyophilization solution (Table 3). The preferred concentration of the cryoprotectant in the precursor solution is described as % w / v of the biomaterial concentration. The concentration ranges of trehalose, hydroxypropyl-β-cyclodextrin, PVP, and dextran can be about 0.1% w / v to about 5% w / v, about 0.1% w / v to about 5% w / v, about 0.1% w / v to about 10% w / v, and about 0.1% w / v to about 10% w / v, respectively.

[0164] These cryoprotectant components can be mixed with the untreated and pre-treated precursor solutions for 15 minutes to 3 hours. The precursor solution can contain a photoinitiator, or the photoinitiator can be added after the precursor solution has been allowed to stand for 15 minutes to 3 hours. In one embodiment, the precursor solution containing the cryoprotectant and the photoinitiator is then irradiated as described elsewhere herein to form photocrosslinked self-degrading bioderived microspheres encapsulating the enzyme and the cryoprotectant. These microspheres can be dehydrated to obtain dry particles with a moisture content of about 10% by mass or lower, about 8% by mass or lower, about 6% by mass or lower, about 4% by mass or lower, about 3% by mass or lower, preferably about 2% by mass or lower, and more preferably about 1% by mass or lower. Under dry conditions sealed in vials, the dry particles can be further sterilized, such as by γ or electron beam radiation as described elsewhere herein. The dehydrated and sterilized enzyme-loaded divalent metal ion crosslinked bioderived microspheres can be stored for ≤24 months.

[0165] Additional Embodiments

[0166] The concentration of the biomaterial can also affect the pore size and robustness of the bivalent-complexed enzyme-loaded bioderived particles. For the preparation of rapidly (>20 minutes to <4 hours) and slowly degrading (5 days to 30 days) enzyme-loaded bivalent metal ion-complexed bioderived particles, the concentration of the biomaterial can be about 0.05% weight / volume (w / v), 0.10% w / v, 0.15% w / v, 0.20% w / v, 0.25% w / v, 0.30% w / v, 0.35% w / v, 0.40% w / v, 0.45% w / v, 0.50% w / v, 0.60% w / v, 0.70% w / v, 0.80% w / v, 0.90% w / v, 1.0% w / v, 1.25% w / v, 1.5% w / v, 1.75% w / v, 2.0% w / v, 2.25% w / v, 2.5% w / v, 2.75% w / v, 3.0% w / v, 3.25% w / v, 3.5% w / v, 3.75% w / v, 4% w / v, 4.25% w / v, 4.5% w / v, 4.75% w / v, 5.0% w / v, 5.25% w / v, 5.5% w / v, 5.75% w / v, 6.0% w / v or greater than about 6.0% w / v.

[0167] In addition, the gelling time of the enzyme-loaded bioderived particles during crosslinking in the metal ion bath can also affect the size, sphericity, and physical robustness of the bivalent metal ion-complexed bioderived particles. Generally, the term "sphericity" can refer to a measure of how closely the shape of an object resembles the shape of a perfect sphere. The roundness of an injectable substance can be important, for example, because an abnormally shaped substance may have difficulty traveling through blood vessels, leading to blood vessel blockage and thus blocking blood flow to various parts of the body. The gelling time can be less than about 1 minute, less than about 2 minutes, less than about 3 minutes, less than about 4 minutes, less than about 5 minutes, less than about 6 minutes, less than about 7 minutes, less than about 8 minutes, less than about 9 minutes, less than about 10 minutes, less than about 11 minutes, less than about 12 minutes, less than about 13 minutes, less than about 14 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, or less than about 30 minutes.

[0168] To enable the enzyme-loaded bioderived particles to achieve a desired degradation period, the amount of enzyme mixed with the biomaterial can be varied. The amount of enzyme mixed with the biomaterial varies between <1 unit to 50 units / mL of biomaterial for the preparation of rapidly (>20 minutes to <4 hours), moderately (5 days to 30 days), or slowly (>30 days) degrading bivalent metal ion-complexed bioderived particles. For clarity, in enzymology, 1 unit (U) is the amount of enzyme that catalyzes the reaction of 1 μmol of substrate per minute. The amount of enzyme loaded into the bivalent metal ion-complexed bioderived particles also depends on the molecular weight or viscosity of the biomaterial.

[0169] In addition, the degradation of enzyme-loaded bioderived particles can also be controlled by modulating enzyme activity. To control the catalytic degradation activity of bioderived particles, the enzyme can be complexed or pretreated with <1 mM of Cu 2+ , Zn 2+ , and Fe 3+ metal ions. These metal ions can inhibit the enzyme activity by approximately 90%. Other metal ions (e.g., Mg 2+ and Ca 2+ ) at 1 mM concentration reduce the activity by 20% to 50%, respectively. Free or unbound metal ions can be removed from the solution by dialysis. These metal ions can inhibit the enzyme activity and can be considered harmful to the enzyme (Inoue et al., “Functional identification of alginate lyase from the brown alga Saccharina japonica”, Sci. Rep. 2019; 9:1-11). In contrast, the same concept is employed in certain embodiments of the present disclosure to modulate the degradation of enzyme-loaded bioderived particles. Under in vivo conditions, the enzyme activity can be modulated using only these metal ions to achieve rapid (>20 minutes to <4 hours) and longer (5 days to 30 days or >30 days) duration degradation of the particles.

[0170] Generally, an enzyme can be immobilized onto an inert or insoluble matrix. This provides resistance to physiological factors (e.g., pH or temperature) that affect the enzyme reaction and also increases the reaction rate. It also keeps the enzyme localized at a certain position (e.g., inside the particle, surface modification, etc.). In certain embodiments of the present disclosure, a modified or native enzyme is immobilized / encapsulated in a reactive biomaterial substrate (not an inert matrix). Thus, another important aspect is to avoid initial degradation during the manufacture of enzyme-loaded bioderived particles from an enzyme-biomaterial precursor solution. To overcome this problem, the following methods are proposed for certain embodiments of the present disclosure.

[0171] The enzyme can be pretreated with metal ion inhibitors (e.g., Cu 2+ , Zn 2+ , Fe 3+ , Mg 2+ , and Ca 2+ ). These metal ions at an optimal concentration that does not affect the physical robustness of the particles can reduce the degradation of the particles by partially inhibiting the enzyme activity.

[0172] Another method is to reduce the temperature of the enzyme-biomaterial precursor solution from ambient temperature to a temperature within the range of 4 - 10 °C. This will reduce or stop the catalytic activity of the enzyme, thus preventing the degradation of the biomaterial. Additionally, the temperature of the divalent metal ion gelling bath can also be reduced to the range of 1 - 10 °C. This metal ion bath is used to gel the droplets of the enzyme-biomaterial solution to form divalent metal ion complexed enzyme-loaded bioderived particles.

[0173] The catalytic activity of the enzyme can also be regulated by changing the pH of the enzyme-biomaterial and gelling bath solutions. The catalytic activity of the alginate lyase used in the present disclosure is optimal in the pH range of 6.8 - 7.5 (see, for example, Farrés et al., “Formation kinetics and rheology of alginate fluid gels produced by in-situ calcium release”, Food Hydrocolloids 40 (2014): 76 - 84). To prevent the initial degradation of the biomaterial during the preparation of the enzyme-loaded bioderived particles, the pH of the enzyme-biomaterial solution can be reduced to 3.0. For this process, a buffer with an ionic strength of < 1 M, preferably < 0.1 M, and most preferably < 0.01 M is used, where the pH range is 3.7 to 5.6. In one embodiment, the buffer is a sodium acetate - acetic acid buffer. Additionally, sodium hydroxide (> 1 M to < 0.01 M) or hydrochloric acid (> 1 M to < 0.01 M) can also be used to achieve the desired pH of the solution (pH 6.5 - 3.0). This results in a reduction or cessation of the enzyme catalytic activity. This regulation of catalytic activity can be attributed to the unfolding of the 3D conformation of the enzyme. By exposing the enzyme-loaded bioderived particles to an aqueous environment with a pH of 6.5 - 7.5, the terminated catalytic activity of the enzyme can be reversed / activated. The preferred buffer for reversing the activity of the enzyme of the present disclosure is a phosphate buffer. At 20 °C, the preferred ionic strength of the phosphate buffer is 0.01 M, where the pH range is 6.5 to 7.5. Sodium hydroxide (> 1 M to < 0.01 M) or hydrochloric acid (> 1 M to < 0.01 M) can also be used to achieve the desired pH of the solution (pH 6.5 - 7.5). Additionally, saline, deionized water, or an aqueous solution with a pH between 6.5 - 7.5 can also be used.

[0174] Therefore, a combination of the above methods can be effectively used to encapsulate or load the enzyme into bioderived particles complexed / gelled with divalent metal ions without degrading the bioderived matrix.

[0175] Under appropriate conditions (low temperature and pH), the proenzyme-biomaterial solution needs to gel in a divalent metal ion bath containing one or more cryoprotectants to crosslink the resulting enzyme-loaded bioderived particles. The composition and conditions of the gelling bath are important for preparing the desired enzyme-loaded bioderived particles. The divalent metal ion component of the gelling bath composition can be selected from Cu 2+ 、Ba 2+ 、Sr 2+ 、Ca 2+ 、Co 2+ 、Ni 2+ 、Mn 2+ and Mg 2+ (Lee et al., "Alginate: properties and biomedical applications," Progress in polymer science 37, no. 1 (2012): 106-126; and Brus et al., "Structure and dynamics of alginate gels cross-linked by polyvalent ions probed via solid state NMR spectroscopy," Biomacromolecules 18, no. 8 (2017): 2478-2488). The divalent cation selection can also affect the crosslinking of the bioderived particles. The binding strength of divalent metal ions to alginate is given in decreasing order of Cu 2+ >Ba 2+ >Sr 2+ >Ca 2+ >Co 2+ >Ni 2+ >Mn 2+ >Mg 2+ , where the preferred metal cations for alginate are Ba 2+ and Ca 2+ . These metal ions can be used at different concentrations in the range of 0.1% w / v to 10% w / v. Adding cryoprotectants to the gelling bath is important in four aspects: (a) it helps to maintain the sphericity and mechanical robustness of the enzyme-loaded bioderived particles during particle dehydration, and (b) when freeze-drying technology is used for dehydration, it also maintains the 3D conformation of the enzyme during the extremely low temperatures and freeze-thaw cycles used during freeze-drying, thus maintaining enzyme activity, (c) it prevents the formation of large ice crystals and helps to maintain the microsphere matrix as well as the bulk (containing cryoprotectant and microspheres) during the freeze-drying process, and (d) it reduces the reconstitution of the freeze-dried product in an aqueous medium without generating bubbling or trapped air bubbles.

[0176] In many cases, it has been observed that when lyophilizing enzymes without the addition of cryoprotectants / cryopreservation media, the residual activity of the enzymes is significantly reduced (Tamiya et al., "Freeze denaturation of enzymes and its prevention with additives," Cryobiology 22, no. 5 (1985): 446-456; and Porter et al., "Effects of freezing on particulate enzymes of rat liver," J. Biol. Chem 205 (1953): 883-891). Cryoprotectant components can include those known in the art, such as sucrose, glycerol, ethylene glycol, sorbitol, trehalose, propylene glycol, or patented / commercially available cryoprotectants. When these cryoprotectants are added to the gelling bath, they are encapsulated or uniformly distributed in the matrix of the bio-derived particles (Chan et al., "Effects of starch filler on the physical properties of lyophilized calcium-alginate beads and the viability of encapsulated cells," Carbohydrate polymers 83, no. 1 (2011): 225-232).

[0177] Alternatively, cryoprotectants can also be used in a post-treatment stage rather than being added to the gelling bath containing divalent metal ions during the manufacturing process of these particles. For example, when preparing lyophilized enzyme-loaded bio-derived particles, cryoprotectants can be used in this post-treatment step. In this process, droplets of the precursor enzyme-biomaterial solution are added to a gelling bath containing only divalent metal ions to form enzyme-loaded bio-derived particles. After separating these particles from the gelling bath, they can be immersed in a suitable cryoprotectant and subjected to a freeze-drying process. Under freeze-drying conditions, the cryoprotectant prevents freeze-denaturation of the enzyme and, by filling the pores formed due to the sublimation of water from the matrix, prevents the collapse of the gel structure, providing defect-free enzyme-loaded bio-derived particles. The particle size can be >40 μm, <200 μm but <2000 μm. In addition, this method can be used to prepare enzyme-loaded bio-derived particles of different morphologies, such as microfibers, core-shell particles, Janus particles, or capsules.

[0178] Furthermore, in certain embodiments, the present disclosure provides the preparation of further enzyme-loaded bio-derived particles further loaded with radiopaque and drugs. To achieve this, it is proposed to use a gelling bath containing Ca2+ Compositions of ions and X-ray imaging metal ions (such as barium, gadolinium, and tantalum metal ions) (Yu et al., "Metal-based X-ray contrast media," Chemical Reviews 99, no. 9 (1999): 2353-2378). Another proposed method is to reconstitute enzyme-loaded bioderived particles with commercially available radiopaque reagents, which are temporarily absorbed into the matrix when the bioderived particles swell in an aqueous medium. The proposed method of loading drugs / biologically active agents (anticancer and osteogenic) into enzyme-loaded bioderived particles involves exposing the particles to the drug for 2-3 hours. The in situ degradation mechanism of enzyme-loaded bioderived particles facilitates drug delivery in the body.

[0179] In certain embodiments, enzyme-loaded bioderived microspheres can be stored for an extended period of time. In certain embodiments, metal ion-complexed enzymes are immobilized into their substrates. It is expected that slow degradation of the matrix begins during storage. This degradation can be stopped by suspending the microspheres in a pH below 5.5. In addition to reducing the operating temperature to below 10 °C to prevent the degradation of alginate microspheres, an alternative method is to freeze or vacuum dry the microspheres. In certain embodiments, the dried microspheres can be loaded into specially designed syringes.

[0180] Figure 19Describe a proposed design for a syringe for reconstituting and / or administering the microspheres of the present disclosure. The syringe can include a plunger 701, which can be in a locked or unlocked position. The syringe can also contain a first chamber containing a suspension medium 702 and a second chamber containing dry microspheres 703. Generally, the syringe can be constructed and arranged such that the contents of each chamber of the syringe are separated (e.g., fluidly separated) until pressure is applied to the plunger, thereby mixing the contents of each chamber (e.g., reconstituting the microspheres). It is contemplated that any multi-chamber lyophilized syringe known in the art can be used. In certain embodiments, the syringe can include a frangible membrane 704 separating the first chamber 701 and the second chamber 702. When pressure is applied to the plunger 701, the frangible membrane is ruptured, and the contents of the first chamber contact the dry microspheres of the second chamber 703 to reconstitute the microspheres. In other embodiments, the syringe includes a liquid bypass conduit. In yet another embodiment, the barrier separating the first and second chambers of the syringe can include a one-way valve. When pressure is applied to the plunger 701, the one-way valve is forced open, and the contents of the first chamber contact the dry microspheres of the second chamber to reconstitute the microspheres. In some embodiments, the reconstitution medium can be water for injection (WFI), saline, or a buffer with a pH in the acidic, basic, or neutral range. Once reconstituted, the microspheres can be used immediately. The syringe can include a quick connector 705 (e.g., a Luer lock connector) for connecting tubing or the like to administer the reconstituted microspheres to a subject.

[0181] Subjects

[0182] A patient treated by any of the methods or compositions described herein can be of any age and can be an adult, infant, or child. In some cases, the patient is 0, 1, 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, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 years old, or within a range therein (e.g., between 2 and 20 years old, between 20 and 40 years old, or between 40 and 90 years old). The patient can be a human or non-human subject.

[0183] Any composition disclosed herein can be administered to non-human subjects, such as laboratory or farm animals. Non-limiting examples of non-human subjects include laboratory or research animals, dogs, goats, guinea pigs, hamsters, mice, pigs, non-human primates (such as gorillas, apes, orangutans, lemurs or baboons), rats, sheep or cattle.

[0184] Additives and Excipients

[0185] In some cases, the enzyme-loaded bio-derived particles or microspheres described herein may contain excipients, which can provide long-term preservation, fill formulations containing active ingredients, facilitate drug absorption, reduce viscosity or enhance the solubility of the bio-derived particles or microspheres. The enzyme-loaded bio-derived particles or microspheres of the present disclosure may contain about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or greater than about 50% excipients by weight or volume.

[0186] In certain embodiments, the enzyme-loaded bio-derived particles or microspheres of the present disclosure may contain one or more solubilizers. As used herein, "solubilizer" includes compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium dioctyl sulfosuccinate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropyl cyclodextrin, ethanol, n-butanol, isopropanol, cholesterol, bile salts, polyethylene glycol 200-600, tetrahydrofurfuryl polyethylene glycol ether, carbitol, propylene glycol, dimethyl isosorbide, etc. The enzyme-loaded bio-derived particles or microspheres of the present disclosure may contain about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or greater than about 50% solubilizer by weight or volume.

[0187] In some embodiments, the compositions described herein include other medicinal or pharmaceutical reagents, carriers, adjuvants, such as preservatives, stabilizers, wetting agents or emulsifiers, solubilizing agents and salts for adjusting the osmotic pressure, osmolality and / or molality of the enzyme-loaded bio-derived particles or microspheres. In some embodiments, the composition contains a stabilizer. In some embodiments, the stabilizer is selected from, for example, fatty acids, fatty alcohols, alcohols, long-chain fatty acid esters, long-chain ethers, hydrophilic derivatives of fatty acids, polyvinylpyrrolidone, polyvinyl ethers, polyvinyl alcohols, hydrocarbons, hydrophobic polymers, hygroscopic polymers and combinations thereof. In some embodiments, amide analogs of stabilizers are also used.

[0188] In some embodiments, the composition comprises a suspending agent. Available suspending agents include (by way of example only) compounds such as polyvinylpyrrolidone (e.g., polyvinylpyrrolidone Kl2, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30), vinylpyrrolidone / vinyl acetate copolymer (S630), polyethylene glycol (e.g., the molecular weight of polyethylene glycol can be about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400), sodium carboxymethyl cellulose, methylcellulose, hydroxypropyl methylcellulose, hydroxymethylcellulose acetate stearate, polysorbate-80, hydroxyethyl cellulose, sodium alginate, gums (e.g., tragacanth and gum arabic, guar gum, xanthan gums, including xanthan gum), sugars, cellulose (e.g., sodium carboxymethyl cellulose, methylcellulose, sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose), polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone, and the like.

[0189] In some embodiments, the composition comprises additional surfactants (cosurfactants) and / or buffers and / or solvents. In some embodiments, the surfactant and / or buffer and / or solvent are a) natural and synthetic lipophilic agents such as phospholipids, cholesterol, and cholesterol fatty acid esters and their derivatives; b) nonionic surfactants which include, for example, polyoxyethylene fatty alcohol esters, sorbitan fatty acid esters (Span), polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene (20) sorbitan monooleate (Tween 80), polyoxyethylene (20) sorbitan monostearate (Tween 60), polyoxyethylene (20) sorbitan monolaurate (Tween 20), and other Tweens), sorbitan esters, glycerol esters such as Myrj and glyceryl triacetate (triacetin), polyethylene glycol, cetyl alcohol, cetostearyl alcohol, stearyl alcohol, polysorbate 80, poloxamer, poloxamine, polyoxyethylene castor oil derivatives (e.g., RH40, Cremphor A25, Cremphor A20, EL, and other Cremophor), sulfosuccinates, alkyl sulfates (SLS); PEG glycerol fatty acid esters such as PEG-8 caprylic / capric glyceride (Labrasol), PEG-4 caprylic / capric glyceride (Labrafac Hydro WL 1219), PEG-32 lauric glyceride (Gelucire 444 / 14), PEG-6 monoolein (Labrafil M1944CS), PEG-6 linolein (Labrafil M 2125CS); propylene glycol mono- and di-fatty acid esters such as propylene glycol laurate, propylene glycol caprylate / caprate; 700, ascorbyl-6-palmitate, stearylamine, sodium lauryl sulfate, polyoxyethylene glyceryl trioleate, and any combination or mixture thereof; c) anionic surfactants including but not limited to calcium carboxymethylcellulose, sodium carboxymethylcellulose, sodium sulfosuccinate, dioctyl, sodium alginate, alkyl polyoxyethylene sulfate, sodium lauryl sulfate, triethanolamine stearate, potassium laurate, bile salts, and any combination or mixture thereof; and d) cationic surfactants such as quaternary compounds, benzalkonium chloride, cetyltrimethylammonium bromide, and lauryldimethylbenzylammonium chloride. The solvent to be used can be selected according to the subject matter under consideration.

[0190] In some embodiments, the compositions disclosed herein include preservatives. Suitable preservatives for the compositions described herein include but are not limited to benzoic acid, boric acid, parabens, phenol, chlorinated phenolic compounds, alcohols, quaternary compounds, quaternary ammonium compounds (such as benzalkonium chloride, cetyltrimethylammonium bromide, or cetylpyridinium chloride), stabilized chlorine dioxide, mercurials (such as merfen or thimerosal), or mixtures thereof.

[0191] Methods of Using Enzyme - Loaded Biologically - Derived Particles

[0192] In another aspect, the present invention provides a method for inducing self-degrading embolization in a subject in need thereof, comprising administering a plurality of microspheres as described elsewhere herein into a blood vessel of the subject. In one embodiment, the blood vessel is the geniculate artery. In one embodiment, the method induces prostate artery embolization, uterine artery embolization, or, when the enzyme-loaded bio-derived particles are mixed with or contain a chemotherapeutic agent, transarterial chemoembolization (TACE).

[0193] In yet another aspect, the present disclosure provides a method for treating a disease or disorder in a subject in need thereof, comprising administering a plurality of microspheres as described elsewhere herein to the subject. In one embodiment, the microspheres are injected at the site of the disease or disorder of the subject. In one embodiment, the microspheres are injected at the site of pain or discomfort in the subject caused by the disease or disorder. In one embodiment, the disease or disorder is tendinopathy. In one embodiment, the disease or condition is selected from osteoarthritis, frozen shoulder, tennis elbow (lateral epicondylitis), golfer's elbow (medial epicondylopathy), pitcher's elbow (flexor tendinitis), Achilles tendinopathy, plantar fasciitis, symptomatic accessory navicular pain, hamstring tendinopathy, jumper's knee (patellar tendinitis), runner's knee (patellofemoral pain syndrome (PFPS)), pes anserine bursitis (knee pain), posterior tibial tendon disease, wrist (TFCC - triangular fibrocartilage complex) tendon disease, trigger finger (stenosing flexor tenosynovitis), and hemarthrosis.

[0194] In another aspect, the present disclosure provides a method for rapidly degrading microspheres in a subject, comprising administering to the subject an emergency rescue solution, wherein a plurality of the microspheres described elsewhere herein have been previously administered to the subject, and the emergency rescue solution comprises an enzyme, an anion, a phosphate buffer, or a combination thereof. In one embodiment, the enzyme is capable of degrading the microspheres and / or wherein the anion degrades the microspheres by chelating one or more metals in the microspheres. In one embodiment, the subject has previously been administered the microspheres disclosed herein, the microspheres comprising alginate particles encapsulating an alginate lyase, and the emergency rescue solution comprises an alginate lyase sugar, optionally in combination with a divalent metal chelator. In another embodiment, the subject has previously been administered the microspheres disclosed herein, the microspheres comprising alginate particles encapsulating an alginate lyase, and the emergency rescue solution comprises citrate.

[0195] In yet another aspect, the present disclosure provides a method for rapidly degrading microspheres in a subject, comprising administering to the subject an emergency rescue solution, wherein a plurality of microspheres comprising a crosslinked biomaterial and lacking an enzyme have been previously administered to the subject. In one embodiment, the microspheres comprising the biomaterial are crosslinked with divalent metal ions. Exemplary divalent metal ions are described elsewhere herein. In one embodiment, the emergency rescue solution comprises an enzyme capable of degrading the biomaterial and an anion or a chelator. In one embodiment, the enzyme is complementary to the biomaterial used to make the microspheres. In one embodiment, the microspheres comprise alginate particles and the enzyme is an alginate lyase, the microspheres comprise pectin particles and the enzyme is pectinase, the microspheres comprise hyaluronic acid particles and the enzyme is hyaluronidase, the microspheres comprise gelatin particles and the enzyme is a matrix metalloproteinase or a protease, the microspheres comprise albumin particles and the enzyme is a peptidase, the microspheres comprise collagen particles and the enzyme is a protease, the microspheres comprise fibrinogen particles and the enzyme is plasmin, the microspheres comprise fibroin particles and the enzyme is a protease, the microspheres comprise starch particles and the enzyme is amylase, the microspheres comprise chitosan particles and the enzyme is chitosanase or lysozyme, the microspheres comprise agar / agarose particles and the enzyme is agarase, the microspheres comprise carrageenan particles and the enzyme is carrageenase, the microspheres comprise pullulan particles and the enzyme is pullulanase, the microspheres comprise dextran particles and the enzyme is dextranase, the microspheres comprise β-glucan particles and the enzyme is β-glucanase, the microspheres comprise cellulose particles and the enzyme is cellulase, or the microspheres comprise lignin particles and the enzyme is ligninase.

[0196] Kits

[0197] In another aspect, the present disclosure provides a kit.

[0198] In one embodiment, the kit comprises the enzyme-loaded bio-derived particles described elsewhere herein. In one embodiment, the particles are microspheres. In one embodiment, the enzyme-loaded bio-derived particles are photocrosslinked. In another embodiment, the enzyme-loaded bio-derived particles are crosslinked using divalent metal ions, heterobifunctional crosslinkers or homobifunctional crosslinkers. In yet another embodiment, the enzyme-loaded bio-derived particles are gelled using a thermal gelation method. In one embodiment, the kit comprises a solvent in which the particles or microspheres of the present disclosure are reconstituted, dissolved, suspended or dispersed. In another embodiment, the kit does not comprise the enzyme-loaded bio-derived particles.

[0199] In one embodiment, the kit comprises one or more components to form an "emergency relief" solution that provides rapid degradation of the particles or microspheres of the present disclosure in an emergency. In one embodiment, one component of the emergency relief solution comprises an enzyme that can act on the particles or microspheres of the present disclosure to degrade them. In one embodiment, the enzyme is a complementary enzyme to the biomaterial used to make the bio-derived particles. Thus, in embodiments where alginate is used as the biomaterial to prepare the bio-derived particles, the enzyme used to form the emergency relief solution is alginate lyase. In embodiments where one component of the emergency relief solution comprises an enzyme, the kit may further comprise a divalent or trivalent metal chelator. In another embodiment, one component of the emergency relief solution comprises an inorganic salt. In one embodiment, the inorganic salt releases an anion when dissolved to form the emergency relief solution, where the anion can chelate metals that may be present in the particles or microspheres, thereby rapidly dissolving the particles or microspheres. In one embodiment, the inorganic salt releases citrate. In one embodiment, the enzyme-loaded bio-derived particles are alginate particles loaded with alginate lyase containing calcium, where anions such as citrate can chelate calcium, resulting in rapid dissolution of the particles. In another embodiment, one component of the emergency relief solution comprises an inorganic salt that releases phosphate and / or sodium ions when dissolved to form the emergency relief solution. In one embodiment, the inorganic salt comprises components that constitute phosphate buffered saline when dissolved to form the emergency relief solution. In one embodiment, the kit includes a solvent in which the components of the emergency relief solution are reconstituted, dissolved, suspended or dispersed.

[0200] In one embodiment, the kit includes instructions. In one embodiment, the instructions provide information on how to reconstitute, dissolve, suspend, or disperse the particles or microspheres of the present disclosure in a solvent to form a solution. In one embodiment, the instructions provide information on how to administer the particle or microsphere solution to a subject in need. In one embodiment, the kit includes a syringe for administering the solution or the particles or microspheres. In one embodiment, the instructions provide information on the dosage of the particles or microspheres that should be administered to a subject in need. In one embodiment, the instructions provide information on when an emergency rescue solution should be administered to a subject and / or how to administer the emergency rescue solution to the subject. In one embodiment, the instructions provide information on how to reconstitute, dissolve, suspend, or disperse one or more components of the emergency rescue solution in a solvent to form a solution. In one embodiment, the kit includes a syringe for administering the emergency rescue solution.

[0201] Other Embodiments and Equivalents

[0202] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0203] It should be understood that the methods described herein are not limited to the specific methods, protocols, subjects, and sequencing techniques described herein and may thus vary. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the methods and compositions described herein, which are limited only by the appended claims. Although some embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, changes, and substitutions will occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed in practicing the present disclosure. It is intended that the appended claims define the scope of the present disclosure and thereby cover methods and structures within the scope of these claims and their equivalents.

[0204] For illustration, reference is made to example applications to describe several aspects. Unless otherwise specified, any implementation may be combined with any other implementation. It should be understood that numerous specific details, relationships, and methods are set forth to provide a comprehensive understanding of the features described herein. However, one of ordinary skill in the art will readily recognize that the features described herein may be practiced without one or more of the specific details or in other ways. The features described herein are not limited by the order of acts or events recited, as some acts may occur in a different order and / or concurrently with other acts or events. In addition, not all acts or events recited are necessary to implement a method in accordance with the features described herein. Moreover, to the extent that the methods of the present disclosure do not depend on a particular order of the steps set forth herein, the particular order of the steps should not be construed as a limitation on the claims. Any claim directed to a method of the present disclosure should not be limited to performing its steps in the order written, and one of ordinary skill in the art can readily understand that the steps can be varied and still remain within the spirit and scope of the present disclosure.

[0205] Although some implementations have been shown and described herein, it will be apparent to those skilled in the art that such implementations are provided by way of example only. The implementations of the present disclosure are not intended to be limited by the specific examples provided within the specification. Although certain implementations of the present disclosure have been described with reference to the foregoing specification, the description and illustration of the implementations herein are not to be construed in a limiting sense. Many variations, changes, and substitutions will occur to those skilled in the art without departing from the present disclosure.

[0206] In addition, it should be understood that all aspects of the implementations of the present disclosure are not limited to the specific depictions, configurations, or relative proportions set forth herein, which depend on various conditions and variables. It should be understood that various alternatives of the implementations of the present disclosure described herein may be employed in practicing the present invention. Accordingly, it is contemplated that the present disclosure will also cover any such alternatives, modifications, variations, or equivalents. It is intended that the appended claims at least in part define the scope of the present invention and thus cover methods and structures within the scope of these claims and their equivalents.

[0207] Those skilled in the art will understand that changes may be made to the exemplary embodiments shown and described above without departing from the broad inventive concept of the present invention. Accordingly, it should be understood that the present disclosure is not limited to the exemplary embodiments shown and described, but is intended to cover modifications within the spirit and scope of the present disclosure as defined by the claims. For example, the specific features of the exemplary embodiments may or may not be part of the claimed invention, and the various features of the disclosed embodiments may be combined. The words "right", "left", "lower" and "upper" designate directions in the accompanying drawings to which reference is made. The words "inward" and "outward" respectively refer to directions toward and away from the geometric center of the fluid delivery device. Unless specifically stated herein, the terms "a", "an" and "the" are not limited to one element, but should be understood to mean "at least one".

[0208] The ranges described herein should be understood as shorthand for all values within the range, including the recited endpoints. For example, a range of 1-50 should be understood to include any number, combination of numbers, or sub-range consisting of 1, 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 and 50.

[0209] It should be understood that at least some of the figures and descriptions of the present disclosure have been simplified to focus on elements relevant to a clear understanding of the present disclosure, while eliminating other elements that those of ordinary skill in the art will understand may also form part of the present disclosure for the sake of clarity. However, since such elements are well known in the art and since they do not necessarily facilitate a better understanding of the present disclosure, no description of such elements is provided herein.

[0210] The following clauses describe certain embodiments of the present disclosure.

[0211] Clause 1. An alginate microsphere capable of self-degrading upon rehydration, comprising:

[0212] An alginate lyase pretreated with a varying temperature, a varying pH, and / or a metal ion enzyme inhibitor;

[0213] An alginate molecule having one or both of the following: (i) a predetermined molecular weight, and (ii) a predetermined ratio of β-D-mannuronic acid (M) blocks to α-L-guluronic acid (G) blocks; and

[0214] A divalent metal ion that crosslinks the alginate molecule,

[0215] Wherein the alginate microspheres are substantially free of water and / or sterilized.

[0216] Clause 2. The alginate microspheres according to Clause 1, wherein the degradation of the alginate microspheres is controlled by one or more of the following: pretreatment with the alginate lyase, the amount of the alginate enzyme in the microspheres, the predetermined molecular weight of the alginate molecule, and the predetermined ratio of the M:G block of the alginate molecule, as well as the composition of the gelling bath, including the amount and / or charge of one or more ions in the gelling bath.

[0217] Clause 3. The alginate microspheres according to Clause 1 or 2, wherein the pH of the alginate lyase in the precursor solution containing the alginate lyase and alginate is in the range of pH 3.0 - 6.4 to prevent the degradation of the alginate, and then crosslinked with a divalent metal cation.

[0218] Clause 4. The alginate microspheres according to any one of Clauses 1 - 3, wherein the metal ion enzyme inhibitor is a reversible inhibitor selected from Cu 2+ 、Zn 2+ and Fe 3+ (e.g., to control the degradation of the alginate in the precursor solution and then crosslink with a divalent metal cation).

[0219] Clause 5. The alginate microspheres according to any one of Clauses 1 - 4, wherein the temperature of the precursor solution is in the range of 1 - 4 °C to control the degradation of the alginate, and then crosslinked with a divalent metal cation.

[0220] Clause 6. The alginate microspheres according to any one of Clauses 1 - 5, wherein the pretreatment of the alginate enzyme in the precursor solution allows a predetermined amount of the enzyme (measured in units U) to be mixed with the alginate molecule.

[0221] Clause 7. The alginate microspheres according to any one of Clauses 1 - 6, wherein the activity of the alginate lyase is adjusted by regulating one or more of the pH of the gelling bath, the temperature of the gelling bath, and the amount of the metal ion enzyme inhibitor in the alginate microspheres.

[0222] Clause 8. The alginate microspheres according to any one of Clauses 1 - 7, wherein the degradation of the alginate microspheres is controlled by the predetermined molecular weight of the alginate molecule.

[0223] Clause 9. The alginate microspheres according to any one of Clauses 1 - 8, wherein the predetermined molecular weight of the alginate molecule is in the range of greater than about 100 kDa to less than about 800 kDa.

[0224] Clause 10. The alginate microspheres according to any one of Clauses 1 - 9, wherein the predetermined ratio of the M:G block controls the degradation of the alginate microspheres.

[0225] Clause 11. The alginate microspheres described in any one of Clauses 1-10, wherein the predetermined ratio of the M:G block is about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, about 90:10 or about 95:5.

[0226] Clause 12. The alginate microspheres described in Clause 11, wherein the alginate microspheres degrade within a period of less than about 5 days.

[0227] Clause 13. The alginate microspheres described in Clause 11 or 12, wherein the alginate microspheres degrade within a period of more than about 2 days.

[0228] Clause 14. The alginate microspheres described in any one of Clauses 1-10, wherein the predetermined ratio of the M:G block is about 50:50, about 45:55, about 40:60, about 35:65, about 30:70, about 25:75, about 20:80, about 15:85, about 10:90 or about 5:95.

[0229] Clause 15. The alginate microspheres described in Clause 14, wherein the alginate microspheres degrade within a period between about 5 days and about 30 days.

[0230] Clause 16. The alginate microspheres described in any one of Clauses 1-15, wherein the pretreated alginate lyase is mixed in the precursor solution having an enzyme activity in the range of 0.025 U / mg to 1 U / mg of alginate.

[0231] Clause 17. The alginate microspheres described in any one of Clauses 1-16, wherein the activity of the alginate lyase is between about 0.05 mU (milliunit) and about 2.5 mU / microsphere.

[0232] Clause 18. The alginate microspheres described in Clause 17, wherein the alginate microspheres degrade within a period of less than about 5 days.

[0233] Clause 19. The alginate microspheres described in any one of Clauses 1-16, wherein the activity of the alginate lyase is between about 0.05 nU (nanounit) and about 0.05 mU / microsphere.

[0234] Clause 20. The alginate microspheres described in Clause 19, wherein the alginate microspheres degrade within a period between about 5 days and about 30 days.

[0235] Clause 21. The alginate microspheres described in any one of Clauses 1-20, which further contain a bioactive agent.

[0236] Clause 22. The alginate microspheres described in Clause 21, wherein the bioactive agent comprises an anti-inflammatory agent and / or an anesthetic to relieve pain associated with embolism in a subject.

[0237] Clause 23. The alginate microspheres described in Clause 21, wherein the bioactive agent comprises an anti-cancer agent or an anti-angiogenic agent.

[0238] Clause 24. The alginate microspheres described in Clause 22, wherein the anti-inflammatory agent comprises hyaluronic acid having a molecular weight between about 1 million (M) and about 5 M Daltons.

[0239] Clause 25. The alginate microspheres described in Clause 24, wherein the ratio of hyaluronic acid to the alginate molecules by weight is about 1:20.

[0240] Clause 26. The alginate microspheres described in any one of Clauses 1-25, which further comprise a cryoprotectant selected from the following: hydroxypropyl-β-cyclodextrin, trehalose, polyvinylpyrrolidone (PVP 40 kDa) of 40 kDa, and dextran (molecular weight of 70 kDa).

[0241] Clause 27. The alginate microspheres described in any one of Clauses 1-26, wherein the alginate microspheres are lyophilized.

[0242] Clause 28. The alginate microspheres described in Clause 27, wherein the residual water content of the lyophilized alginate microspheres is in the range of about 1% to about 3% by mass.

[0243] Clause 29. The alginate microspheres described in any one of Clauses 1-28, wherein the sphericity of the alginate microspheres is at least about 0.7, at least about 0.75, at least about 0.8, at least about 0.85, at least about 0.9, at least about 0.95, or at least about 0.99.

[0244] Clause 30. The alginate microspheres described in any one of Clauses 1-29, wherein the alginate microspheres or the lyophilized alginate microspheres are sterilized.

[0245] Clause 31. The alginate microspheres described in Clause 30, wherein the sterilization comprises high-energy radiation sterilization, gamma-ray sterilization, or electron beam sterilization.

[0246] Clause 32. The alginate microspheres described in Clause 31, wherein the sterilization comprises gamma radiation from a cobalt 60 isotope between about 15 and about 25 kGy according to ISO 11137-1:2006 or electron beam radiation of about 25 kGy.

[0247] Clause 33. The alginate microspheres according to any one of Clauses 1-31, wherein when stored at a given temperature, the shelf life of the alginate microspheres is at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 36 months, at least about 48 months or at least about 60 months.

[0248] Clause 34. The alginate microspheres according to Clause 33, wherein the given temperature is between about 2 °C and about 8 °C.

[0249] Clause 35. The alginate microspheres according to Clause 33, wherein the given temperature is about room temperature (RT).

[0250] Clause 36. The alginate microspheres according to any one of Clauses 27-35, wherein the alginate microspheres are reconstituted in saline or a radiopaque contrast agent in saline at physiological pH.

[0251] Clause 37. A method for preparing alginate microspheres capable of self-degrading upon rehydration, the method comprising:

[0252] forming droplets from a precursor solution, the precursor solution comprising: an alginate lyase pretreated by a changing temperature, by a changing pH and / or with a metal ion enzyme inhibitor; and an alginate molecule having one or both of the following: (a) a predetermined molecular weight, and (b) a predetermined ratio of β-D-mannuronic acid (M) blocks to α-L-guluronic acid (G) blocks;

[0253] contacting the droplets with a gelling bath comprising a cryoprotectant and a divalent metal ion, thereby crosslinking the alginate molecules to form alginate microspheres; and

[0254] dehydrating and optionally sterilizing the alginate microspheres, thereby substantially removing water from the microspheres.

[0255] Clause 38. The method according to Clause 37, wherein the precursor solution comprises one or more cryoprotectants.

[0256] Clause 39. The method according to Clause 37 or 38, wherein the gelling bath comprises one or more cryoprotectants.

[0257] Clause 40. The method according to Clause 38 or 39, wherein the cryoprotectant is selected from hydroxypropyl-β-cyclodextrin, trehalose, polyvinylpyrrolidone (PVP 40 kDa) of 40 kDa and dextran (molecular weight of 70 kDa).

[0258] Clause 41. The method according to Clause 40, wherein the concentration of trehalose in the precursor solution is from about 0.1% w / v to about 20% w / v.

[0259] Clause 42. The method according to clause 40 or 41, wherein the concentration of hydroxypropyl-β-cyclodextrin is from about 0.1% w / v to about 2% w / v.

[0260] Clause 43. The method according to any one of clauses 40 - 42, wherein the concentration of PVP 40 kDa in the precursor solution is from about 0.1% w / v to about 1% w / v.

[0261] Clause 44. The method according to any one of clauses 40 - 43, wherein the concentration of dextran (molecular weight 70 kDa) in the precursor solution is from about 0.1% w / v to about 1% w / v.

[0262] Clause 45. The method according to any one of clauses 39 - 44, wherein the precursor solution and the gelling bath contain the same cryoprotectant.

[0263] Clause 46. The method according to clause 45, wherein the precursor solution and the gelling bath contain equal or approximately equal concentrations of the same cryoprotectant.

[0264] Clause 47. The method according to any one of clauses 37 - 46, wherein the dehydration comprises lyophilizing the alginate microspheres.

[0265] Clause 48. The method according to clause 47, wherein the residual water content of the lyophilized alginate microspheres is in the range of about 1% to about 3% by mass.

[0266] Clause 49. The method according to any one of clauses 37 - 48, wherein the sphericity of the alginate microspheres is at least about 0.7, at least about 0.75, at least about 0.8, at least about 0.85, at least about 0.9, at least about 0.95 or at least about 0.99.

[0267] Clause 50. The method according to any one of clauses 37 - 49, further comprising sterilizing the alginate microspheres or the lyophilized alginate microspheres.

[0268] Clause 51. The method according to clause 50, wherein the sterilization comprises high-energy radiation sterilization, gamma-ray sterilization or electron beam sterilization.

[0269] Clause 52. The method according to clause 51, wherein the sterilization comprises gamma radiation from a cobalt 60 isotope between about 15 and about 25 kGy according to ISO 11137 - 1:2006 or electron beam radiation of about 25 kGy.

[0270] Clause 53. The method according to any one of Clauses 37 - 51, further comprising storing the alginate microspheres for at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 36 months, at least about 48 months, or at least about 60 months when stored at a given temperature.

[0271] Clause 54. The method according to any one of Clauses 37 - 53, wherein when stored at a given temperature, the shelf life of the alginate microspheres is at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 36 months, at least about 48 months, or at least about 60 months.

[0272] Clause 55. The method according to Clause 53 or 54, wherein the given temperature is between about 2°C and about 8°C.

[0273] Clause 56. The method according to Clause 53 or 54, wherein the given temperature is about room temperature (RT).

[0274] Clause 57. The method according to any one of Clauses 37 - 56, further comprising administering the alginate microspheres, the lyophilized alginate microspheres, or the sterilized microspheres to a subject.

[0275] Clause 58. The method according to Clause 57, wherein the step of administering the alginate microspheres, the lyophilized alginate microspheres, or the sterilized microspheres to the subject is carried out by reconstituting the lyophilized alginate microspheres or the sterilized microspheres in saline or a radiopaque contrast agent at physiological pH.

[0276] Clause 59. The method according to any one of Clauses 37 - 58, wherein the formation of the droplets is carried out using a method selected from: droplet casting, spray freezing / spray cooling, spray drying, microfluidic droplet generation, and jet cutting.

[0277] Clause 60. The method according to any one of Clauses 37 - 59, wherein the degradation of the alginate microspheres is controlled by one or more of: pretreatment with alginate lyase, the amount of alginate enzyme in the microspheres, the predetermined molecular weight of the alginate molecule, and the predetermined ratio of the M:G blocks of the alginate molecule, as well as the composition of the gelling bath, including the amount and / or charge of one or more ions in the gelling bath.

[0278] Clause 61. The method according to any one of Clauses 37 - 60, wherein the pH of the alginate lyase in the precursor solution containing alginate lyase and alginate is in the range of pH 3.0 - 6.4 to prevent degradation of the alginate, followed by crosslinking with divalent metal cations.

[0279] Clause 62. The method according to any one of Clauses 37 - 60, wherein the metal ion enzyme inhibitor is selected from Cu 2+ , Zn 2+ and Fe 3+ and is a reversible inhibitor (e.g., to control the degradation of the alginate in the precursor solution and then crosslink with divalent metal cations).

[0280] Clause 63. The method according to any one of Clauses 37 - 62, wherein the temperature of the precursor solution is in the range of 1 - 4 °C to control the degradation of the alginate and then crosslink with divalent metal cations.

[0281] Clause 64. The method according to any one of Clauses 37 - 62, wherein the pretreatment of the alginate enzyme in the precursor solution allows a predetermined amount of enzyme (measured in units U) to be mixed with the alginate molecules.

[0282] Clause 65. The method according to any one of Clauses 37 - 64, wherein the activity of the alginate lyase is adjusted by regulating one or more of the pH of the gelling bath, the temperature of the gelling bath, and the amount of the metal ion enzyme inhibitor in the alginate microspheres.

[0283] Clause 66. The method according to any one of Clauses 37 - 64, wherein the degradation of the alginate microspheres is controlled by a predetermined molecular weight of the alginate molecules.

[0284] Clause 67. The method according to any one of Clauses 37 - 66, wherein the predetermined molecular weight of the alginate molecules is in the range of greater than about 100 kDa to less than about 800 kDa.

[0285] Clause 68. The method according to any one of Clauses 37 - 67, wherein the predetermined ratio of the M:G block controls the degradation of the alginate microspheres.

[0286] Clause 69. The method according to any one of Clauses 37 - 68, wherein the predetermined ratio of the M:G block is about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, about 90:10, or about 95:5.

[0287] Clause 70. The method according to Clause 69, wherein the alginate microspheres degrade within a period of less than about 5 days.

[0288] Clause 71. The method according to Clause 69 or 70, wherein the alginate microspheres degrade within a period of greater than about 2 days.

[0289] Clause 72. The method according to any one of Clauses 37 - 68, wherein the predetermined ratio of the M:G block is about 50:50, about 45:55, about 40:60, about 35:65, about 30:70, about 25:75, about 20:80, about 15:85, about 10:90 or about 5:95.

[0290] Clause 73. The method according to Clause 72, wherein the alginate microspheres degrade within a time period between about 5 days and about 30 days.

[0291] Clause 74. The method according to any one of Clauses 37 - 73, wherein the pretreated alginate lyase is mixed in the precursor solution having an enzyme activity in the range of 0.0025 U / mg to 1 U / mg of alginate.

[0292] Clause 75. The method according to any one of Clauses 37 - 74, wherein the pretreated alginate lyase is mixed in the precursor solution having an enzyme activity in the range of 0.125 U / mg to 0.250 U / mg of alginate.

[0293] Clause 76. The method according to Clause 75, wherein the alginate microspheres degrade within a time period of less than about 5 days.

[0294] Clause 77. The method according to any one of Clauses 37 - 74, wherein the pretreated alginate lyase is mixed in the precursor solution having an enzyme activity in the range of 0.025 U / mg to 0.125 U / mg of alginate.

[0295] Clause 78. The method according to Clause 77, wherein the alginate microspheres degrade within a time period between about 5 days and about 30 days.

[0296] Clause 79. The method according to any one of Clauses 37 - 74, wherein the pretreated alginate lyase is mixed in the precursor solution having an enzyme activity in the range of 0.0025 U / mg to 0.005 U / mg of alginate.

[0297] Clause 80. The method according to Clause 79, wherein the alginate microspheres degrade within a time period greater than about 30 days.

[0298] Clause 81. The method according to any one of Clauses 37 - 80, wherein the precursor solution and / or the gelling bath further comprises a bioactive agent.

[0299] Clause 82. The method according to Clause 81, wherein the bioactive agent comprises an anti - inflammatory agent and / or an anesthetic agent to relieve pain associated with embolism in a subject.

[0300] Clause 83. The method according to Clause 81, wherein the bioactive agent comprises an anti-cancer agent or an anti-angiogenic agent.

[0301] Clause 84. The method according to Clause 83, wherein the anti-inflammatory agent comprises hyaluronic acid having a molecular weight between about 1 million (M) and about 5 M Daltons.

[0302] Clause 85. The method according to Clause 84, wherein the ratio of hyaluronic acid to the alginate molecules by weight is about 1:20.

[0303] Clause 86. The method according to any one of Clauses 37 - 85, wherein the pH of the gelling bath is less than about 6.5.

[0304] Clause 87. The method according to any one of Clauses 37 - 85, wherein the pH of the gelling bath is equal to or approximately equal to the pH of the precursor solution.

[0305] Clause 88. The method according to any one of Clauses 37 - 87, wherein the temperature of the precursor solution is equal to or approximately equal to between 1 °C and about 4 °C.

[0306] Clause 89. A photo-polymerizable alginate microsphere capable of self-degrading upon rehydration, comprising:

[0307] An alginate lyase pretreated by varying temperature, by varying pH and / or with a metal ion enzyme inhibitor;

[0308] Alginate molecules functionalized with ethylenically unsaturated functional groups, said molecules having one or both of the following: (i) a predetermined molecular weight, and (ii) a predetermined ratio of β-D-mannuronic acid (M) blocks to α-L-guluronic acid (G) blocks; and

[0309] A photoinitiator,

[0310] Wherein the alginate molecules are crosslinked by irradiating the photoinitiator, and

[0311] Wherein the alginate microspheres are substantially free of water and / or are sterilized.

[0312] Clause 90. The alginate microsphere according to Clause 89, wherein the ethylenically unsaturated functional group is selected from acrylate, methacrylate, vinyl and allyl.

[0313] Clause 91. The alginate microsphere according to Clause 89 or 90, wherein the degradation of the alginate microspheres is controlled by one or more of the following: the pretreatment of the alginate lyase, the predetermined molecular weight of the alginate molecules, and the predetermined ratio of M:G blocks of the alginate molecules, as well as the composition of the gelling bath, including the amount and / or charge of one or more ions in the gelling bath.

[0314] Clause 92. The alginate microspheres described in any one of Clauses 89 - 91, wherein the pH of the alginate lyase in the precursor solution containing the alginate lyase and alginate is in the range of pH 3.0 - 6.4 to prevent degradation of the alginate, and then crosslinked with a divalent metal cation.

[0315] Clause 93. The alginate microspheres described in any one of Clauses 89 - 91, wherein the metal ion enzyme inhibitor is selected from Cu 2+ 、Zn 2+ and Fe 3+ and is a reversible inhibitor (e.g., to control degradation of the alginate in the precursor solution, and then crosslinked with a divalent metal cation).

[0316] Clause 94. The alginate microspheres described in any one of Clauses 89 - 93, wherein the temperature of the precursor solution is in the range of 1 - 4 °C to control degradation of the alginate, and then crosslinked with a divalent metal cation.

[0317] Clause 95. The alginate microspheres described in any one of Clauses 89 - 93, wherein the pretreatment of the alginate enzyme in the precursor solution allows a predetermined amount of enzyme (measured in units U) to be mixed with the alginate molecules.

[0318] Clause 96. The alginate microspheres described in any one of Clauses 89 - 95, wherein the activity of the alginate lyase is adjusted by regulating one or more of the pH of the gelling bath, the temperature of the gelling bath, and the amount of the metal ion enzyme inhibitor in the alginate microspheres.

[0319] Clause 97. The alginate microspheres described in any one of Clauses 89 - 96, wherein the degradation of the alginate microspheres is controlled by a predetermined molecular weight of the alginate molecules.

[0320] Clause 98. The alginate microspheres described in any one of Clauses 89 - 96, wherein the predetermined molecular weight of the alginate molecules is in the range of greater than about 100 kDa to less than about 800 kDa.

[0321] Clause 99. The alginate microspheres described in any one of Clauses 89 - 98, wherein the predetermined ratio of the M:G block controls the degradation of the alginate microspheres.

[0322] Clause 100. The alginate microspheres described in any one of Clauses 89 - 99, wherein the predetermined ratio of the M:G block is about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, about 90:10 or about 95:5.

[0323] Clause 101. The alginate microspheres described in Clause 100, wherein the alginate microspheres degrade within a time period of less than about 5 days.

[0324] Clause 102. The alginate microspheres described in Clause 100 or 101, wherein the alginate microspheres degrade within a time period of greater than about 2 days.

[0325] Clause 103. The alginate microspheres described in any one of Clauses 89 - 99, wherein the predetermined ratio of the M:G block is about 50:50, about 45:55, about 40:60, about 35:65, about 30:70, about 25:75, about 20:80, about 15:85, about 10:90 or about 5:95.

[0326] Clause 104. The alginate microspheres described in Clause 103, wherein the alginate microspheres degrade within a time period between about 5 days and about 30 days.

[0327] Clause 105. The alginate microspheres described in any one of Clauses 89 - 104, wherein the pretreated alginate lyase is mixed in the precursor solution having an enzyme activity in the range of 0.025 U / mg to 1 U / mg alginate.

[0328] Clause 106. The alginate microspheres described in any one of Clauses 89 - 105, wherein the activity of the alginate lyase is between about 0.05 mU (milliunit) and about 2.5 mU / microsphere.

[0329] Clause 107. The alginate microspheres described in Clause 106, wherein the alginate microspheres degrade within a time period of less than about 5 days.

[0330] Clause 108. The alginate microspheres described in any one of Clauses 89 - 105, wherein the activity of the alginate lyase is between about 0.05 nU (nanounit) and about 0.05 mU / microsphere.

[0331] Clause 109. The alginate microspheres described in Clause 108, wherein the alginate microspheres degrade within a time period between about 5 days and about 30 days.

[0332] Clause 110. The alginate microspheres described in any one of Clauses 89 - 109, which further comprise a bioactive agent.

[0333] Clause 111. The alginate microspheres described in Clause 110, wherein the bioactive agent comprises an anti - inflammatory agent to relieve pain associated with embolism in a subject.

[0334] Clause 112. The alginate microspheres described in Clause 111, wherein the anti - inflammatory agent comprises hyaluronic acid having a molecular weight between about 1 million (M) and about 5 M daltons.

[0335] Clause 113. The alginate microspheres described in Clause 112, wherein the ratio of hyaluronic acid to the alginate molecules is about 1:20 by weight.

[0336] Clause 114. The alginate microspheres according to any one of Clauses 89 - 113, further comprising a cryoprotectant selected from: hydroxypropyl-β-cyclodextrin, trehalose, polyvinylpyrrolidone (PVP 40 kDa) with a molecular weight of 40 kDa, and dextran (with a molecular weight of 70 kDa).

[0337] Clause 115. The alginate microspheres according to any one of Clauses 89 - 114, wherein the alginate microspheres are lyophilized.

[0338] Clause 116. The alginate microspheres described in Clause 103, wherein the residual water content of the lyophilized alginate microspheres is in the range of about 1% to about 3% by mass.

[0339] Clause 117. The alginate microspheres according to any one of Clauses 89 - 116, wherein the sphericity of the alginate microspheres is at least about 0.7, at least about 0.75, at least about 0.8, at least about 0.85, at least about 0.9, at least about 0.95, or at least about 0.99.

[0340] Clause 118. The alginate microspheres according to any one of Clauses 89 - 117, wherein the alginate microspheres or the lyophilized alginate microspheres are sterilized.

[0341] Clause 119. The alginate microspheres described in Clause 118, wherein the sterilization comprises high-energy radiation sterilization, gamma-ray sterilization, or electron beam sterilization.

[0342] Clause 120. The alginate microspheres described in Clause 119, wherein the sterilization comprises gamma radiation from cobalt-60 isotope between about 15 to about 25 kGy according to ISO 11137-1:2006 or electron beam radiation of about 25 kGy.

[0343] Clause 121. The alginate microspheres according to any one of Clauses 89 - 120, wherein when stored at a given temperature, the shelf life of the alginate microspheres is at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 36 months, at least about 48 months, or at least about 60 months.

[0344] Clause 122. The alginate microspheres described in Clause 121, wherein the given temperature is between about 2°C and about 8°C.

[0345] Clause 123. The alginate microspheres described in Clause 121, wherein the given temperature is about room temperature (RT).

[0346] Clause 124. The alginate microspheres according to any one of Clauses 115 - 123, wherein the alginate microspheres are reconstituted in saline or a radiopaque contrast agent in saline at physiological pH.

[0347] Clause 125. A method for preparing a photopolymerizable alginate microsphere capable of self - degrading upon rehydration, the method comprising:

[0348] Forming droplets from a precursor solution, the precursor solution comprising: an alginate lyase pretreated by varying temperature, by varying pH, and / or with a metal ion enzyme inhibitor; an alginate molecule functionalized with an ethylenically unsaturated functional group, the molecule having one or both of the following: (a) a predetermined molecular weight, and (b) a predetermined ratio of β - D - mannuronic acid (M) blocks to α - L - guluronic acid (G) blocks; and a photoinitiator;

[0349] Irradiating the droplets comprising the photoinitiator so that the alginate molecules cross - link to form photopolymerized alginate microspheres; and

[0350] Dehydrating and optionally sterilizing the alginate microspheres to substantially remove water from the microspheres.

[0351] Clause 126. The method according to Clause 125, wherein the ethylenically unsaturated functional group is selected from acrylate, methacrylate, vinyl, and allyl.

[0352] Clause 127. The method according to Clause 125 or 126, wherein the precursor solution comprises one or more cryoprotectants.

[0353] Clause 128. The method according to any one of Clauses 125 - 127, wherein the gelling bath comprises one or more cryoprotectants.

[0354] Clause 129. The method according to Clause 127 or 128, wherein the cryoprotectant is selected from hydroxypropyl - β - cyclodextrin, trehalose, polyvinylpyrrolidone (PVP 40 kDa), and dextran (molecular weight 70 kDa).

[0355] Clause 130. The method according to Clause 129, wherein the concentration of trehalose in the precursor solution is from about 0.1% w / v to about 20% w / v.

[0356] Clause 131. The method according to Clause 129 or 130, wherein the concentration of hydroxypropyl - β - cyclodextrin is from about 0.1% w / v to about 2% w / v.

[0357] Clause 132. The method according to any one of Clauses 129 - 131, wherein the concentration of PVP 40 kDa in the precursor solution is from about 0.1% w / v to about 1% w / v.

[0358] Clause 133. The method according to any one of Clauses 129 - 132, wherein the concentration of dextran (molecular weight 70 kDa) in the precursor solution is from about 0.1% w / v to about 1% w / v.

[0359] Clause 134. The method according to any one of Clauses 128 - 133, wherein the precursor solution and the gelling bath contain the same cryoprotectant.

[0360] Clause 135. The method according to Clause 134, wherein the precursor solution and the gelling bath contain equal or approximately equal concentrations of the same cryoprotectant.

[0361] Clause 136. The method according to any one of Clauses 125 - 135, wherein the dehydration comprises freeze - drying the alginate microspheres.

[0362] Clause 137. The method according to Clause 136, wherein the residual water content of the freeze - dried alginate microspheres is in the range of about 1% to about 3% by mass.

[0363] Clause 138. The method according to any one of Clauses 125 - 137, wherein the sphericity of the alginate microspheres is at least about 0.7, at least about 0.75, at least about 0.8, at least about 0.85, at least about 0.9, at least about 0.95 or at least about 0.99.

[0364] Clause 139. The method according to any one of Clauses 125 - 138, which further comprises sterilizing the alginate microspheres or the freeze - dried alginate microspheres.

[0365] Clause 140. The method according to Clause 139, wherein the sterilization comprises high - energy radiation sterilization, gamma - ray sterilization or electron - beam sterilization.

[0366] Clause 141. The alginate microspheres according to Clause 140, wherein the sterilization comprises gamma radiation from a cobalt 60 isotope between about 15 and about 25 kGy according to ISO 11137 - 1:2006 or electron - beam radiation of about 25 kGy.

[0367] Clause 142. The method according to any one of Clauses 125 - 141, which further comprises storing the alginate microspheres for at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 36 months, at least about 48 months or at least about 60 months when stored at a given temperature.

[0368] Clause 143. The method according to any one of Clauses 125 - 142, wherein when stored at a given temperature, the shelf life of the alginate microspheres is at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 36 months, at least about 48 months, or at least about 60 months.

[0369] Clause 144. The method according to Clause 142 or 143, wherein the given temperature is between about 2°C and about 8°C.

[0370] Clause 145. The method according to Clause 142 or 143, wherein the given temperature is about room temperature (RT).

[0371] Clause 146. The method according to any one of Clauses 125 - 145, further comprising administering the alginate microspheres, the lyophilized alginate microspheres, or the sterilized microspheres to a subject.

[0372] Clause 147. The method according to Clause 146, wherein the step of administering the alginate microspheres, the lyophilized alginate microspheres, or the sterilized microspheres to a subject is carried out by reconstituting the alginate microspheres, the lyophilized alginate microspheres, or the sterilized microspheres with saline or a radiopaque contrast agent in saline at physiological pH.

[0373] Clause 148. The method according to any one of Clauses 125 - 147, wherein the method of causing the precursor solution to flow through the pores to form droplets is selected from: droplet casting, spray freezing / spray cooling, spray drying, and microfluidic droplet generation.

[0374] Clause 149. The method according to any one of Clauses 125 - 148, wherein the degradation of the alginate microspheres is controlled by one or more of: pretreatment with alginate lyase, a predetermined molecular weight of the alginate molecule, and a predetermined ratio of the M:G blocks of the alginate molecule, as well as the composition of the gelling bath, including the amount and / or charge of one or more ions in the gelling bath.

[0375] Clause 150. The method according to any one of Clauses 125 - 149, wherein the pH of the alginate lyase in the precursor solution containing alginate lyase and alginate is in the range of pH 3.0 - 6.4 to prevent degradation of the alginate, and then crosslinked with divalent metal cations.

[0376] Clause 151. The method according to any one of Clauses 125 - 150, wherein the metal ion enzyme inhibitor is selected from Cu 2+ 、Zn 2+ and Fe 3+Reversible inhibitors (e.g., to control the degradation of the alginate in the precursor solution and then crosslink with divalent metal cations).

[0377] Clause 152. The method according to any one of Clauses 125 - 151, wherein the temperature of the precursor solution is in the range of 1 - 4 °C to control the degradation of the alginate and then crosslink with divalent metal cations.

[0378] Clause 153. The method according to any one of Clauses 125 - 152, wherein the pretreatment of the alginate enzyme in the precursor solution allows the enzyme with the desired activity (referred to as unit U) to be mixed with the alginate that controls the degradation of the alginate microspheres for a certain length of time.

[0379] Clause 154. The method according to any one of Clauses 125 - 153, wherein the degradation of the alginate microspheres is controlled by the predetermined molecular weight of the alginate molecules.

[0380] Clause 155. The method according to any one of Clauses 125 - 154, wherein the predetermined molecular weight of the alginate molecules is in the range of greater than about 100 kDa to less than about 800 kDa.

[0381] Clause 156. The method according to any one of Clauses 125 - 155, wherein the predetermined ratio of the M:G block controls the degradation of the alginate microspheres.

[0382] Clause 157. The method according to any one of Clauses 125 - 156, wherein the predetermined ratio of the M:G block is about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, about 90:10 or about 95:5.

[0383] Clause 158. The method according to Clause 157, wherein the alginate microspheres degrade within a period of less than about 5 days.

[0384] Clause 159. The method according to Clause 157 or 158, wherein the alginate microspheres degrade within a period of greater than about 2 days.

[0385] Clause 160. The method according to any one of Clauses 125 - 156, wherein the predetermined ratio of the M:G block is about 50:50, about 45:55, about 40:60, about 35:65, about 30:70, about 25:75, about 20:80, about 15:85, about 10:90 or about 5:95.

[0386] Clause 161. The method according to Clause 160, wherein the alginate microspheres degrade within a period between about 5 days and about 30 days.

[0387] Clause 162. The method according to any one of Clauses 125 - 161, wherein the pretreated alginate lyase is mixed in the precursor solution having an enzyme activity in the range of 0.0025 U / mg to 1 U / mg of alginate.

[0388] Clause 163. The method according to any one of Clauses 125 - 162, wherein the pretreated alginate lyase is mixed in the precursor solution having an enzyme activity in the range of 0.125 U / mg to 0.250 U / mg of alginate.

[0389] Clause 164. The method according to Clause 163, wherein the alginate microspheres degrade within a period of less than about 5 days.

[0390] Clause 165. The method according to any one of Clauses 125 - 162, wherein the pretreated alginate lyase is mixed in the precursor solution having an enzyme activity in the range of 0.025 U / mg to 0.125 U / mg of alginate.

[0391] Clause 166. The method according to Clause 165, wherein the alginate microspheres degrade within a period between about 5 days and about 30 days.

[0392] Clause 167. The method according to any one of Clauses 124 - 147, wherein the pretreated alginate lyase is mixed in the precursor solution having an enzyme activity in the range of 0.0025 U / mg to 0.005 U / mg of alginate.

[0393] Clause 168. The method according to Clause 167, wherein the alginate microspheres degrade within a period greater than about 30 days.

[0394] Clause 169. The method according to any one of Clauses 125 - 168, wherein the precursor solution and / or the gelling bath further comprises a bioactive agent.

[0395] Clause 170. The method according to Clause 169, wherein the bioactive agent comprises an anti - inflammatory agent to relieve pain associated with embolism in a subject.

[0396] Clause 171. The method according to Clause 170, wherein the anti - inflammatory agent comprises hyaluronic acid having a molecular weight between about 1 million (M) and about 5 M Daltons.

[0397] Clause 172. The method according to Clause 171, wherein the ratio of hyaluronic acid to the alginate molecules by weight is about 1:20.

[0398] Clause 173. The method according to any one of Clauses 125 - 172, wherein the pH of the gelling bath is less than about 6.5.

[0399] Clause 174. A method according to any one of Clauses 125 - 172, wherein the pH of the coagulation bath is equal to or approximately equal to the pH of the precursor solution.

[0400] Clause 175. A method according to any one of Clauses 125 - 174, wherein the temperature of the precursor solution is equal to or approximately equal to between about 1 °C and about 4 °C.

[0401] Clause 176. A method of inducing self - degrading embolisms in a subject in need thereof, comprising administering into a blood vessel of the subject a plurality of alginate microspheres according to any one of Clauses 1 - 36 and 89 - 124.

[0402] Clause 177. The method according to Clause 176, wherein the blood vessel is the geniculate artery.

[0403] Clause 178. A syringe, comprising:

[0404] A first chamber containing alginate microspheres according to any one of Clauses 1 - 36 or 89 - 124;

[0405] A second chamber axially disposed with respect to the first chamber, the second chamber containing a reconstitution medium; and

[0406] A plunger configured such that when depressed, the alginate microspheres are exposed to the reconstitution medium, thereby reconstituting the alginate microspheres.

[0407] Clause 179. The syringe according to Clause 178, further comprising a frangible membrane separating the first chamber and the second chamber, wherein when the plunger is depressed, the frangible membrane breaks to expose the alginate microspheres to the reconstitution medium, thereby reconstituting the alginate microspheres.

[0408] Clause 180. Microspheres capable of self - degrading upon rehydration, for administration to a mammalian subject in need thereof, the microspheres comprising:

[0409] A biocompatible polysaccharide material that cannot be enzymatically hydrolyzed by the mammalian subject, wherein the biocompatible polysaccharide material has one or both of the following: (i) a predetermined molecular weight, and (ii) a predetermined ratio of β - D - mannuronic acid (M) blocks to α - L - guluronic acid (G) blocks;

[0410] An enzyme capable of hydrolyzing the biocompatible polysaccharide material, wherein the enzyme is not naturally present in the mammalian subject, and wherein the enzyme is pretreated by varying temperature, by varying pH, and / or with a metal ion enzyme inhibitor; and

[0411] A divalent metal ion that cross - links the biocompatible polysaccharide material,

[0412] wherein said microspheres are substantially free of water and / or are sterilized.

[0413] Clause 181. The microspheres according to clause 180, wherein said biocompatible polysaccharide material comprises alginate.

[0414] Clause 182. The microspheres according to clause 180 or 181, wherein said enzyme comprises alginate lyase.

[0415] Clause 183. The microspheres according to any one of clauses 180-182, wherein said biocompatible polysaccharide material is resorbable.

[0416] Clause 184. The microspheres according to any one of clauses 180-183, wherein said biocompatible polysaccharide material is stable to enzymatic hydrolysis in said mammalian subject.

[0417] Clause 185. The microspheres according to any one of clauses 180-184, wherein the resorption rate of said biocompatible polysaccharide material is more precisely controlled by including an amount of an enzyme that is not found in said mammal and that has a specific effect of causing said embolization material to decompose once in the body.

[0418] Clause 186. A method of preparing microspheres that are capable of self-degrading upon rehydration for administration to a mammalian subject in need thereof, said method comprising:

[0419] forming droplets from a precursor solution comprising: a biocompatible polysaccharide material that is not enzymatically hydrolyzable by said mammalian subject, wherein said biocompatible polysaccharide material has one or both of the following: (i) a predetermined molecular weight, and (ii) a predetermined ratio of β-D-mannuronic acid (M) blocks to α-L-guluronic acid (G) blocks; and an enzyme capable of hydrolyzing said biocompatible polysaccharide material, wherein said enzyme is not naturally present in said mammalian subject, and wherein said enzyme is pretreated by varying temperature, by varying pH, and / or with a metal ion enzyme inhibitor;

[0420] contacting said droplets with a gelling bath comprising a cryoprotectant and a divalent metal ion, thereby crosslinking said biocompatible polysaccharide material to form microspheres,

[0421] dehydrating and optionally sterilizing said microspheres, thereby substantially removing water from said microspheres.

[0422] Clause 187. The method according to clause 186, wherein said biocompatible polysaccharide material comprises alginate.

[0423] Clause 188. The method according to clause 186 or 187, wherein said enzyme comprises alginate lyase.

[0424] Article 189. A method according to any one of Articles 186 - 188, wherein the biocompatible polysaccharide material is resorbable.

[0425] Article 190. A method according to any one of Articles 186 - 189, wherein the biocompatible polysaccharide material is stable against enzymatic hydrolysis in the mammalian subject.

[0426] Article 191. A method according to any one of Articles 186 - 190, wherein the resorption rate of the biocompatible polysaccharide material is more precisely controlled by including an amount of an enzyme that is not found in the mammal and that has a specific action of causing the embolization material to decompose once in the body.

[0427] Article 192. Photopolymerized microspheres that are capable of self - degrading upon rehydration, for administration to a mammalian subject in need thereof, comprising:

[0428] A biocompatible polysaccharide material that cannot be enzymatically hydrolyzed by the mammalian subject, wherein the biocompatible polysaccharide material has one or both of the following: (i) a predetermined molecular weight, and (ii) a predetermined ratio of β - D - mannuronic acid (M) blocks to α - L - guluronic acid (G) blocks;

[0429] An enzyme capable of hydrolyzing the biocompatible polysaccharide material, wherein the enzyme is not naturally present in the mammalian subject, and wherein the enzyme is pretreated by varying temperature, by varying pH, and / or with a metal ion enzyme inhibitor; and

[0430] A photoinitiator,

[0431] wherein the biocompatible polysaccharide material is cross - linked by irradiating the photoinitiator, and

[0432] wherein the microspheres are substantially water - free and / or sterilized.

[0433] Article 193. The microspheres according to Article 192, wherein the biocompatible polysaccharide material comprises alginate.

[0434] Article 194. The microspheres according to Article 192 or 193, wherein the enzyme comprises alginate lyase.

[0435] Article 195. The microspheres according to any one of Articles 192 - 194, wherein the biocompatible polysaccharide material is resorbable.

[0436] Article 196. The microspheres according to any one of Articles 192 - 195, wherein the biocompatible polysaccharide material is stable against enzymatic hydrolysis in the mammalian subject.

[0437] Article 197. The microspheres described in any one of Articles 192 - 196, wherein the resorption rate of the biocompatible polysaccharide material is more precisely controlled by including a certain amount of an enzyme that is not found in the mammal and has a specific effect of causing the embolization material to decompose once in the body.

[0438] Article 198. A method for preparing a photopolymerizable microsphere that is capable of self - degrading upon rehydration for administration to a mammalian subject in need, the method comprising:

[0439] Flowing a precursor solution through pores to form droplets, the precursor solution comprising: a biocompatible polysaccharide material that cannot be enzymatically hydrolyzed by the mammalian subject, wherein the biocompatible polysaccharide material has one or both of the following: (i) a predetermined molecular weight, and (ii) a predetermined ratio of β - D - mannuronic acid (M) blocks to α - L - guluronic acid (G) blocks; an enzyme capable of hydrolyzing the biocompatible polysaccharide material, wherein the enzyme is not naturally present in the mammalian subject, and wherein the enzyme is pretreated by varying temperature, by varying pH, and / or with a metal ion enzyme inhibitor; and a photoinitiator; and

[0440] Irradiating the droplets comprising the photoinitiator so that the biocompatible polysaccharide material cross - links to form a photopolymerized microsphere,

[0441] Dehydrating and optionally sterilizing the microspheres to substantially remove water from the microspheres.

[0442] Article 199. The method according to Article 198, wherein the biocompatible polysaccharide material comprises alginate.

[0443] Article 200. The method according to Article 198 or 199, wherein the enzyme comprises alginate lyase.

[0444] Article 201. The method according to any one of Articles 198 - 200, wherein the biocompatible polysaccharide material is resorbable.

[0445] Article 202. The method according to any one of Articles 198 - 201, wherein the biocompatible polysaccharide material is stable to enzymatic hydrolysis within the mammalian subject.

[0446] Article 203. The method according to any one of Articles 198 - 202, wherein the resorption rate of the biocompatible polysaccharide material is more precisely controlled by including a certain amount of an enzyme that is not found in the mammal and has a specific effect of causing the embolization material to decompose once in the body.

[0447] Article 300. A method for preparing alginate microspheres that are capable of self - degrading upon rehydration, the method comprising:

[0448] Forming droplets from a precursor solution using a microfluidic platform, the precursor solution comprising:

[0449] (i) An alginate lyase pretreated with different ranges of alkaline pH solutions and different temperatures (1 - 4 °C);

[0450] (ii) Alginate molecules having one or both of the following: (a) a predetermined molecular weight, and (b) a predetermined ratio of β-D-mannuronic acid (M) blocks to α-L-guluronic acid (G) blocks;

[0451] (iii) A crosslinking agent, such as Ca-EDTA or CaCO3; and

[0452] (iv) An excipient

[0453] Contacting the droplets with a gelling solution comprising a surfactant, an oil, and acetic acid, thereby crosslinking the alginate molecules to form alginate microspheres; and

[0454] Dehydrating and optionally sterilizing the alginate microspheres, thereby substantially removing water from the microspheres.

[0455] Clause 301. Alginate microspheres prepared by the method described in Clause 300.

[0456] Clause 302. The alginate microspheres described in Clause 301, wherein the degradation of the alginate microspheres is controlled by one or more of the following: the pretreatment of the alginate lyase, the amount of the alginate enzyme in the microspheres, the predetermined molecular weight of the alginate molecules, and the predetermined ratio of the M:G blocks of the alginate molecules, as well as divalent cation-crosslinking.

[0457] Clause 303. The alginate microspheres described in Clause 302, wherein at a temperature of 1 - 4 °C, the pH of the precursor solution containing alginate, alginate lyase, and a crosslinking agent (Ca-EDTA or CaCO3) is in the range of 8 - 13 to prevent the degradation of the alginate and the gelling of the precursor solution.

[0458] Clause 304. The alginate microspheres according to any one of Clauses 301 - 303, wherein the pretreatment of the alginate enzyme in the precursor solution allows a predetermined amount of the enzyme (measured in units U) to be mixed with the alginate molecules.

[0459] Article 305. The alginate microspheres described in any one of Articles 301 - 304, wherein after the droplets of the precursor solution have an alkaline pH, and a low temperature can be generated by a microfluidic chip, and crosslinking is carried out for a duration of 1 minute to 3 hours in a solution containing acetic acid, oil, and surfactant in the range of 0.05% v / v to 5% v / v. Under acidic conditions, due to the ionization of Ca-EDTA or CaCO3 and the encapsulated enzyme remaining inactive under acidic pH conditions, crosslinking of alginate with Ca 2+ ions occurs, thus preventing degradation of the alginate beads.

[0460] Article 306. The alginate microspheres described in any one of Articles 301 - 305, wherein by exposing the beads to calcium chloride of < 10% w / v for a duration of > 1 minute to < 24 hours, the Ca 2+ crosslinked alginate beads containing alginate lyase can be further crosslinked by divalent Ca 2+ cations. The duration of the crosslinking period controls the degradation of alginate particles under physiological conditions.

[0461] Article 307. The alginate microspheres described in Article 306, wherein the calcium chloride solution further contains excipients required for the freeze-drying step.

[0462] Article 308. The alginate microspheres described in Article 307, wherein the Ca 2+ crosslinked alginate beads containing alginate lyase are further washed in an aqueous medium containing excipients to remove unbound Ca 2+ ions.

[0463] Article 309. The alginate microspheres described in Article 308, wherein the Ca 2+ crosslinked alginate beads containing alginate lyase are dispersed in a solution containing excipients and subjected to freeze-drying to produce freeze-dried Ca 2+ crosslinked alginate beads containing alginate lyase.

[0464] Article 310. The alginate microspheres described in Article 309, wherein the freeze-dried Ca 2+ crosslinked alginate beads containing alginate lyase are further subjected to a sterilization step (γ-radiation and electron beam radiation).

[0465] Article 311. The alginate microspheres described in Article 310, wherein the freeze-dried and sterilized Ca 2+ crosslinked alginate beads containing alginate lyase can be reconstituted in an aqueous solution of neutral pH that activates the alginate lyase and initiates degradation of the alginate particles.

[0466] Article 401. Alginate microspheres capable of self - degrading upon rehydration, comprising:

[0467] An alginate lyase pretreated by varying temperature, by varying pH, and / or with a metal ion enzyme inhibitor;

[0468] Alginate molecules having one or both of the following: (i) a predetermined molecular weight, and (ii) a predetermined ratio of β - D - mannuronic acid (M) blocks to α - L - guluronic acid (G) blocks; and

[0469] A divalent metal ion cross - linking the alginate molecules,

[0470] wherein the alginate microspheres are substantially water - free and / or sterilized.

[0471] Article 402. The alginate microspheres according to Article 401, wherein the degradation of the alginate microspheres is controlled by one or more of the following: the pretreatment of the alginate lyase, the amount of the alginate enzyme in the microspheres, the predetermined molecular weight of the alginate molecules, the predetermined ratio of M:G blocks of the alginate molecules, and the composition of the gelling bath, including the amount and / or charge of one or more ions in the gelling bath.

[0472] Article 403. The alginate microspheres according to Article 401, wherein at least one of (i) - (iii) applies:

[0473] (i) The metal ion enzyme inhibitor is a reversible inhibitor selected from Cu 2+ , Zn 2+ and Fe 3+ .

[0474] (ii) The pretreatment of the alginate enzyme in the precursor solution allows a predetermined amount of the enzyme (measured in units U) to be mixed with the alginate molecules, and

[0475] (iii) The activity of the alginate lyase is adjusted by regulating one or more of the pH of the gelling bath, the temperature of the gelling bath, and the amount of the metal ion enzyme inhibitor in the alginate microspheres.

[0476] Article 404. The alginate microspheres according to Article 401, wherein at least one of (i) - (v) applies:

[0477] (i) The predetermined molecular weight of the alginate molecules is in the range of greater than about 100 kDa to less than about 800 kDa,

[0478] (ii) The predetermined ratio of the M:G block is about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, about 90:10 or about 95:5,

[0479] (iii) The predetermined ratio of the M:G block is about 50:50, about 45:55, about 40:60, about 35:65, about 30:70, about 25:75, about 20:80, about 15:85, about 10:90 or about 5:95,

[0480] (iv) The activity of the alginate lyase is between about 0.05 mU (milliunits) and about 2.5 mU / microsphere, and

[0481] (v) The activity of the alginate lyase is between about 0.05 nU (nanounits) and about 0.05 mU / microsphere.

[0482] Clause 405. The alginate microspheres described in Clause 404, where at least one of (a)-(d) applies:

[0483] (a) The alginate microspheres of (ii) degrade within a period of less than about 5 days or greater than about 2 days,

[0484] (b) The alginate microspheres of (iii) degrade within a period between about 5 days and about 30 days,

[0485] (c) The alginate microspheres of (iv) degrade within a period of less than about 5 days, and

[0486] (d) The alginate microspheres of (v) degrade within a period between about 5 days and about 30 days.

[0487] Clause 406. The alginate microspheres described in Clause 401, where at least one of (i)-(vii) applies:

[0488] (i) The microspheres further contain a bioactive agent,

[0489] (ii) The microspheres further contain a cryoprotectant selected from the following: hydroxypropyl-β-cyclodextrin, trehalose, polyvinylpyrrolidone (PVP 40 kDa), dextran (70 kDa molecular weight), glucose, lactose, maltodextrin, mannitol, ethylene glycol, and polyethylene glycol,

[0490] (iii) The alginate microspheres are lyophilized,

[0491] (iv) The sphericity of the alginate microspheres is at least about 0.7, at least about 0.75, at least about 0.8, at least about 0.85, at least about 0.9, at least about 0.95 or at least about 0.99,

[0492] (v) Sterilizing the alginate microspheres, or freeze - drying and sterilizing the alginate microspheres,

[0493] (vi) When stored at a given temperature, the shelf - life of the alginate microspheres is at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 36 months, at least about 48 months or at least about 60 months, and

[0494] (vii) Reconstituting the freeze - dried alginate microspheres in saline or a radiopaque contrast agent in saline at physiological pH.

[0495] Clause 407. The alginate microspheres as described in Clause 406, wherein at least one of (a) - (d) applies:

[0496] (a) The bioactive agent of (i) comprises an anti - inflammatory agent, an anesthetic, an anti - cancer agent or an anti - angiogenic agent,

[0497] (b) The residual water content of the freeze - dried alginate microspheres of (iii) is in the range of about 1% to about 3% by mass,

[0498] (c) The sterilization of (v) comprises high - energy radiation sterilization, gamma - ray sterilization or electron - beam sterilization, and

[0499] (d) The given temperature of (vi) is between about 2°C and about 8°C or about room temperature (RT).

[0500] Clause 408. The alginate microspheres as described in Clause 407, wherein the anti - inflammatory agent of (a) comprises hyaluronic acid having a molecular weight between about 1 million (M) and about 5 M daltons, or the sterilization of (c) comprises gamma - radiation from a cobalt 60 isotope between about 15 and about 25 kGy or electron - beam radiation of about 25 kGy according to ISO 11137 - 1:2006.

[0501] Clause 409. A method for preparing alginate microspheres capable of self - degrading upon rehydration, the method comprising:

[0502] Forming droplets from a precursor solution, the precursor solution comprising:

[0503] Alginate lyase pretreated by varying temperature, by varying pH and / or with a metal ion enzyme inhibitor; and

[0504] Alginate molecules having one or both of the following: (a) a predetermined molecular weight, and (b) a predetermined ratio of β-D-mannuronic acid (M) blocks to α-L-guluronic acid (G) blocks;

[0505] Contacting the droplets with a gelling bath comprising divalent metal ions to crosslink the alginate molecules to form alginate microspheres; and

[0506] Dehydrating and optionally sterilizing the alginate microspheres to substantially remove water from the microspheres.

[0507] The method of clause 410. The method of clause 409, wherein at least one of (i)-(x) applies:

[0508] (i) The precursor solution comprises one or more cryoprotectants,

[0509] (ii) The gelling bath comprises one or more cryoprotectants,

[0510] (iii) The pH of the alginate lyase in the precursor solution containing alginate lyase and alginate is in the range of pH 3.0-6.4,

[0511] (iv) The metal ion enzyme inhibitor is a reversible inhibitor selected from Cu 2+ , Zn 2+ and Fe 3+ of,

[0512] (v) The temperature of the precursor solution is in the range of 1-4 °C,

[0513] (vi) The pretreatment of the alginate enzyme in the precursor solution allows a predetermined amount of enzyme (measured in units U) to be mixed with the alginate molecules,

[0514] (vii) Adjusting the activity of the alginate lyase by adjusting one or more of the pH of the gelling bath, the temperature of the gelling bath, and the amount of the metal ion enzyme inhibitor in the alginate microspheres,

[0515] (viii) The pH of the gelling bath is less than about 6.5,

[0516] (ix) The pH of the gelling bath is equal to or approximately equal to the pH of the precursor solution, and

[0517] (x) The precursor solution and / or the gelling bath further comprises a bioactive agent.

[0518] The method of clause 411. The method of clause 409, wherein at least one of (i)-(iv) applies:

[0519] (i) The dehydration includes freeze-drying the alginate microspheres,

[0520] (ii) The formation of the droplets is carried out using a method selected from: droplet casting, spray freezing / spray cooling, spray drying, microfluidic droplet generation, and jet cutting,

[0521] (iii) The sphericity of the alginate microspheres is at least about 0.7, at least about 0.75, at least about 0.8, at least about 0.85, at least about 0.9, at least about 0.95, or at least about 0.99, and

[0522] (iv) When stored at a given temperature, the shelf life of the alginate microspheres is at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 36 months, at least about 48 months, or at least about 60 months.

[0523] Clause 412. The method according to Clause 410, wherein the cryoprotectants in (i) and (ii) are each independently selected from hydroxypropyl-β-cyclodextrin, trehalose, polyvinylpyrrolidone (PVP 40 kDa), dextran (molecular weight 70 kDa), glucose, lactose, maltodextrin, mannitol, ethylene glycol, and polyethylene glycol.

[0524] Clause 413. The method according to Clause 412, wherein at least one of (a)-(f) applies:

[0525] (a) The concentration of trehalose in the precursor solution (i) is from about 0.1% w / v to about 20% w / v,

[0526] (b) The concentration of hydroxypropyl-β-cyclodextrin in the precursor solution (i) or the gelling bath (ii) is from about 0.1% w / v to about 2% w / v,

[0527] (c) The concentration of PVP 40 kDa in the precursor solution (i) is from about 0.1% w / v to about 1% w / v,

[0528] (d) The concentration of dextran (molecular weight 70 kDa) in the precursor solution (i) is from about 0.1% w / v to about 1% w / v,

[0529] (e) The precursor solution (i) and the gelling bath (ii) contain the same cryoprotectant, and

[0530] (f) The precursor solution (i) and the gelling bath (ii) contain equal or approximately equal concentrations of the same cryoprotectant.

[0531] Clause 414. The method according to Clause 411, wherein the residual water content of the lyophilized alginate microspheres is in the range of about 1% to about 3% by mass.

[0532] Clause 415. The method according to Clause 409, further comprising at least one step selected from (i)-(iv)

[0533] (i) sterilizing the alginate microspheres or the alginate microspheres that have been dehydrated by lyophilization,

[0534] (ii) storing the alginate microspheres for at least about 3 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 36 months, at least about 48 months, or at least about 60 months when stored at a given temperature,

[0535] (iii) administering the alginate microspheres or the alginate microspheres that have been dehydrated by lyophilization to a subject, and

[0536] (iv) reconstructing the alginate microspheres or the alginate microspheres that have been dehydrated by lyophilization using saline or a radiopaque contrast agent in saline at physiological pH.

[0537] Clause 416. The method according to Clause 415, wherein at least one of (a)-(d) applies:

[0538] (a) The sterilization of (i) comprises high-energy radiation sterilization, gamma-ray sterilization, or electron beam sterilization,

[0539] (b) The sterilization of (i) comprises gamma radiation from a cobalt 60 isotope between about 15 and about 25 kGy according to ISO 11137-1:2006 or electron beam radiation of about 25 kGy,

[0540] (c) The given temperature of (ii) is between about 2°C and about 8°C, and

[0541] (d) The given temperature of (ii) is about room temperature (RT).

[0542] Clause 417. The method according to Clause 409, wherein the degradation of the alginate microspheres is controlled by one or more of the following: pretreatment with alginate lyase, the amount of alginate enzyme in the microspheres, the predetermined molecular weight of the alginate molecule, and the predetermined ratio of the M:G block of the alginate molecule, as well as the composition of the gelling bath, including the amount and / or charge of one or more ions in the gelling bath.

[0543] Clause 418. The method according to Clause 409, wherein at least one of (i)-(vii) applies:

[0544] (i) The predetermined molecular weight of the alginate molecule is in the range of greater than about 100 kDa to less than about 800 kDa,

[0545] (ii) The predetermined ratio of the M:G block is about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, about 90:10 or about 95:5,

[0546] (iii) The predetermined ratio of the M:G block is about 50:50, about 45:55, about 40:60, about 35:65, about 30:70, about 25:75, about 20:80, about 15:85, about 10:90 or about 5:95,

[0547] (iv) Mixing the pretreated alginate lyase in the precursor solution having an enzyme activity in the range of 0.0025 U / mg to 1 U / mg alginate,

[0548] (v) Mixing the pretreated alginate lyase in the precursor solution having an enzyme activity in the range of 0.125 U / mg to 0.250 U / mg alginate,

[0549] (vi) Mixing the pretreated alginate lyase in the precursor solution having an enzyme activity in the range of 0.025 U / mg to 0.125 U / mg alginate, and

[0550] (vii) Mixing the pretreated alginate lyase in the precursor solution having an enzyme activity in the range of 0.0025 U / mg to 0.005 U / mg alginate.

[0551] The method according to clause 418 of clause 419, wherein at least one of (a)-(e) applies:

[0552] (a) The alginate microspheres degrade within a period of less than about 5 days or greater than about 2 days,

[0553] (b) The alginate microspheres of (iii) degrade within a period between about 5 days and about 30 days,

[0554] (c) The alginate microspheres of (v) degrade within a period of less than about 5 days,

[0555] (d) The alginate microspheres of (vi) degrade within a period between about 5 days and about 30 days, and

[0556] (e) The alginate microspheres of (vii) degrade within a period greater than about 30 days.

[0557] Article 420. The method of Article 410, wherein the bioactive agent in (x) comprises an anti-inflammatory agent, an anesthetic, an anti-cancer agent, or an anti-angiogenic agent.

[0558] Article 421. The method of Article 420, wherein the anti-inflammatory agent comprises hyaluronic acid having a molecular weight between about 1 million (M) and about 5 M Daltons.

[0559] Article 509. A method for preparing alginate microspheres capable of self-degrading upon rehydration, the method comprising:

[0560] forming droplets from a precursor solution using a microfluidic platform, the precursor solution comprising:

[0561] (i) an alginate lyase pretreated with an alkaline pH and a temperature less than about 15 °C;

[0562] (ii) alginate molecules having one or both of the following: (a) a predetermined molecular weight, and (b) a predetermined ratio of β-D-mannuronic acid (M) blocks to α-L-guluronic acid (G) blocks; and

[0563] (iii) a divalent cation crosslinking agent;

[0564] contacting the droplets with a gelling solution comprising oil and acid, thereby crosslinking the alginate molecules to form alginate microspheres; and

[0565] dehydrating and optionally sterilizing the alginate microspheres, thereby substantially removing water from the microspheres.

[0566] Article 510. The method of Article 509, wherein the degradation of the alginate microspheres is controlled by one or more of the following: the pretreatment of the alginate lyase, the amount of the alginate enzyme in the microspheres, the predetermined molecular weight of the alginate molecules, and the predetermined ratio of M:G blocks of the alginate molecules, and the divalent cation crosslinking of the alginate molecules.

[0567] Article 511. The method of Article 509 or 510, wherein at least one of (i)-(viii) applies:

[0568] (i) pretreating the alginate lyase with a pH of about 8 to about 13,

[0569] (ii) pretreating the alginate lyase with a temperature of about 1 °C to about 4 °C,

[0570] (iii) the pretreatment of the alginate enzyme in the precursor solution allows a predetermined amount of the enzyme (measured in units U) to be mixed with the alginate molecules,

[0571] (iv) The pH of the precursor solution is from about 8 to about 13 and is maintained at a temperature of from about 1 °C to about 4 °C,

[0572] (v) The precursor solution further comprises an excipient,

[0573] (vi) The divalent cation crosslinking agent is Ca released from Ca-EDTA or CaCO3 2+ ,

[0574] (vii) The acid is acetic acid, and

[0575] (viii) The gelling solution further comprises a surfactant.

[0576] Clause 512. The method according to any one of Clauses 509-511, wherein the gelling solution comprises oil, from about 0.05% v / v to about 5% v / v acetic acid and a surfactant, and the droplets are contacted with the gelling solution for from about 1 minute to about 3 hours to crosslink the alginate molecules.

[0577] Clause 513. The method according to any one of Clauses 509-512, wherein the step of dehydrating and optionally sterilizing the alginate microspheres is carried out by crosslinking the alginate microspheres with divalent Ca 2+ ions.

[0578] Clause 514. The method according to any one of Clauses 509-512, wherein the step of dehydrating and optionally sterilizing the alginate microspheres is carried out by further crosslinking the alginate molecules with a second divalent Ca 2+ ion.

[0579] Clause 515. The method according to Clause 514, wherein the alginate molecules are further crosslinked with a second divalent Ca 2+ ion to form alginate microspheres crosslinked with divalent Ca 2+ ions.

[0580] Clause 516. The method according to Clause 513 or 514, wherein the alginate microspheres or alginate molecules are crosslinked by exposure to a solution comprising less than about 10% w / v CaCl2 for from about 1 minute to about 24 hours.

[0581] Clause 517. The method according to Clause 516, wherein the solution further comprises an excipient.

[0582] Clause 518. The method according to Clause 517, wherein the step of dehydrating the alginate microspheres or the divalent Ca 2+ crosslinked alginate microspheres comprises freeze-drying the alginate microspheres or the divalent Ca 2+ crosslinked alginate microspheres.

[0583] Clause 519. The method according to Clause 518, which comprises using gamma radiation and / or electron beam radiation to sterilize the lyophilized alginate microspheres or the lyophilized divalent Ca 2+ crosslinked alginate microspheres.

[0584] Clause 520. The method according to Clause 519, which comprises reconstituting the lyophilized and sterilized alginate microspheres or the lyophilized and sterilized divalent Ca 2+ crosslinked alginate microspheres in an aqueous solution at neutral pH.

[0585] Clause 601. Microspheres capable of self-degrading upon rehydration, comprising:

[0586] an enzyme pretreated by varying temperature, by varying pH, and / or with a metal ion enzyme inhibitor; and

[0587] a crosslinked biomaterial;

[0588] wherein:

[0589] the crosslinked biomaterial forms a bio-derived microsphere encapsulating the enzyme; and

[0590] the microspheres are substantially free of water and / or sterilized.

[0591] Clause 602. The microspheres according to Clause 601, wherein the enzyme is an enzyme that acts on the biomaterial and degrades the biomaterial.

[0592] Clause 603. The microspheres according to Clause 601 or 602, wherein the biomaterial comprises a polysaccharide, a protein, or a glycoprotein.

[0593] Clause 604. The microspheres according to any one of Clauses 601-603, further comprising a photoinitiator, and the biomaterial comprises a photocrosslinkable moiety that is photocrosslinked.

[0594] Clause 605. The microspheres according to any one of Clauses 601-604, wherein the self-degradation of the microspheres is controlled by one or more of the following: the pretreatment of the enzyme, the concentration of the enzyme in the microspheres, the enzyme activity, and the predetermined molecular weight of the biomaterial.

[0595] Clause 606. The microspheres according to any one of Clauses 601-605, wherein the biomaterial is crosslinked by divalent metal ions.

[0596] Clause 607. The microspheres according to Clause 606, wherein the self-degradation of the microspheres is controlled by one or more of the following: the pretreatment of the enzyme, the concentration of the enzyme in the microspheres, the enzyme activity, the predetermined molecular weight of the biomaterial, the divalent metal ions used to crosslink the biomaterial, and the amount of the divalent metal ions used to crosslink the biomaterial.

[0597] Clause 608. The microspheres described in any one of Clauses 601 - 607, wherein the microspheres degrade by themselves within less than about 4 hours, within a time period greater than about 2 days to less than about 5 days, within a time period greater than about 5 days to less than about 30 days, or greater than about 30 days.

[0598] Clause 609. The microspheres described in any one of Clauses 601 - 608, wherein:

[0599] The enzyme activity is between about 0.0075 U / mg and 0.25 U / mg of biomaterial, and the microspheres degrade within a time period greater than about 20 minutes to less than about 4 hours;

[0600] The enzyme activity is between about 0.005 U / mg and about ≥0.0025 U / mg of biomaterial, and the microspheres degrade within a time period greater than about 5 days to less than about 30 days; or

[0601] The enzyme activity is less than about 0.0025 U / mg of biomaterial, and the microspheres degrade within a time period greater than about 30 days.

[0602] Clause 610. The microspheres described in any one of Clauses 601 - 609, wherein the residual water content of the microspheres is between about 1% by mass and about 10% by mass.

[0603] Clause 611. The microspheres described in Clause 610, wherein the microspheres are freeze-dried or dehydrated using supercritical CO2.

[0604] Clause 612. The microspheres described in any one of Clauses 601 - 611, wherein the microspheres are sterilized by γ-radiation at about 6 - 10 kGy.

[0605] Clause 613. The microspheres described in any one of Clauses 601 - 612, wherein the microspheres further comprise an anti-inflammatory agent, a chemotherapeutic agent, an antioxidant, a corticosteroid, or a combination thereof.

[0606] Clause 614. The microspheres described in Clause 613, wherein the microspheres comprise one or more of the following:

[0607] An anti-inflammatory agent selected from hyaluronic acid, ibuprofen, flurbiprofen, meloxicam, nabumetone, etodolac, ketorolac, ketoprofen, diflunisal, naproxen, diclofenac, celecoxib, mefenamic acid, etoricoxib, indomethacin, aspirin, arnica, curcumin, bromelain, and acetaminophen,

[0608] An antioxidant selected from glutathione, α-tocopherol, ergothioneine, N-acetylcysteine, ascorbic acid, vitamin A, vitamin E, allopurinol, melatonin, resveratrol, bucillamine, turmeric, lycopene, lipoic acid, cobalamin, flavonoids, quercetin, ebselen and edaravone; or

[0609] A corticosteroid selected from methylprednisolone, dexamethasone, triamcinolone acetonide, betamethasone, beclomethasone and hydrocortisone.

[0610] Item 615. The microspheres according to any one of Items 601-614, wherein: the biomaterial comprises alginate and the enzyme is alginate lyase, the biomaterial comprises pectin and the enzyme is pectinase, the biomaterial comprises hyaluronic acid and the enzyme is hyaluronidase, the biomaterial comprises gelatin and the enzyme is matrix metalloproteinase or protease, the biomaterial comprises albumin and the enzyme is peptidase, the biomaterial comprises collagen and the enzyme is protease, the biomaterial comprises fibrinogen and the enzyme is plasmin, the biomaterial comprises fibroin and the enzyme is protease, the biomaterial comprises starch and the enzyme is amylase, the biomaterial comprises chitosan and the enzyme is chitosanase or lysozyme, the biomaterial comprises agar / agarose and the enzyme is agarase, the biomaterial comprises carrageenan and the enzyme is carrageenase, the biomaterial comprises pullulan and the enzyme is pullulanase, the biomaterial comprises dextran and the enzyme is dextranase, the biomaterial comprises b-glycan and the enzyme is b-glycanase, the biomaterial comprises cellulose and the enzyme is cellulase, or the biomaterial comprises lignin and the enzyme is ligninase.

[0611] Item 616. The microspheres according to Item 615, wherein the biomaterial comprises alginate and the enzyme is alginate lyase.

[0612] Item 617. The microspheres according to Item 616, wherein the enzyme is pretreated by maintaining the enzyme at a pH of about 3.0 to about 6.4, by maintaining the enzyme at a temperature of about 1 °C to about 4 °C, or by treating the enzyme with a reversible metal ion enzyme inhibitor selected from Cu 2+ 、Zn 2+ and Fe 3+ .

[0613] Item 618. The microspheres according to Item 616 or 617, wherein the biomaterial is selected from Cu 2+ 、Ba 2+ 、Sr 2+ 、Ca 2+ 、Co 2+ 、Ni 2+ 、Mn 2+ and Mg2+ Crosslinked with divalent metal ions.

[0614] Clause 619. The microspheres described in any one of Clauses 616 - 618, wherein:

[0615] The enzyme activity is between about 0.0075 U / mg and 0.25 U / mg alginate, and the microspheres degrade within a period of less than about 4 hours;

[0616] The enzyme activity is between about 0.005 U / mg and about 0.0025 U / mg alginate, and the microspheres degrade within a period between about 5 days and about 30 days; or

[0617] The enzyme activity is less than about 0.0025 U / mg biomaterial, and the microspheres degrade within a period greater than about 30 days.

[0618] Clause 620. A method for preparing microspheres capable of self - degrading upon rehydration, the method comprising:

[0619] Forming droplets from a precursor solution, the precursor solution comprising:

[0620] (i) An enzyme pretreated by varying temperature, by varying pH, and / or with a metal ion enzyme inhibitor; and

[0621] (ii) A biomaterial;

[0622] Contacting the droplets with a gelling bath comprising a cryoprotectant and divalent metal ions, thereby cross - linking the biomaterial to form bio - derived microspheres encapsulating the enzyme; and

[0623] Dehydrating the microspheres and optionally sterilizing, thereby substantially removing water from the microspheres.

[0624] Clause 621. The method according to Clause 620, wherein the precursor solution further comprises one or more cryoprotectants.

[0625] Clause 622. The method according to Clause 620 or 621, wherein each cryoprotectant is independently selected from hydroxypropyl - β - cyclodextrin, trehalose, polyvinylpyrrolidone, and dextran.

[0626] Clause 623. The method according to any one of Clauses 620 - 622, wherein the enzyme is an enzyme that acts on the biomaterial and degrades the biomaterial.

[0627] Clause 624. The method according to any one of Clauses 620 - 623, wherein the biomaterial comprises a polysaccharide, a protein, or a glycoprotein.

[0628] Article 625. The method according to any one of Articles 620 - 624, wherein the microspheres comprise: alginate particles encapsulating alginate lyase, pectin particles encapsulating pectinase, gelatin particles encapsulating matrix metalloproteinase or protease, or carrageenan particles encapsulating carrageenase.

[0629] Article 626. The method according to any one of Articles 620 - 625, wherein the self - degradation of the microspheres is controlled by one or more of the following: pretreatment of the enzyme, the concentration of the enzyme in the microspheres, the enzyme activity, the predetermined molecular weight of the biomaterial, the divalent metal ions used for cross - linking the biomaterial, and the amount of divalent metal ions used for cross - linking the biomaterial.

[0630] Article 627. The method according to any one of Articles 620 - 626, wherein the microspheres self - degrade in less than about 4 hours, in a time period greater than about 2 days to less than about 5 days, in a time period greater than about 5 days to less than about 30 days, or in a time period greater than about 30 days.

[0631] Article 628. The method according to any one of Articles 620 - 627, wherein:

[0632] The enzyme activity is between about 0.0075 U / mg and 0.25 U / mg of biomaterial, and the microspheres degrade in a time period greater than about 20 minutes to less than about 4 hours;

[0633] The enzyme activity is between about 0.005 U / mg and about ≥0.0025 U / mg of biomaterial, and the microspheres degrade in a time period greater than about 5 days to less than about 30 days; or

[0634] The enzyme activity is less than about 0.0025 U / mg of biomaterial, and the microspheres degrade in a time period greater than about 30 days.

[0635] Article 629. The method according to any one of Articles 620 - 628, wherein:

[0636] The droplets are in contact with the gelling bath in the range of 10 minutes to 1 hour, and the resulting microspheres degrade in a time period greater than about 20 minutes to less than about 4 hours;

[0637] The droplets are in contact with the gelling bath in the range of 1 hour to 12 hours, and the resulting microspheres degrade in a time period greater than about 5 days to less than about 30 days; or

[0638] The droplets are in contact with the gelling bath in the range of 12 hours to 24 hours, and the resulting microspheres degrade in a time period greater than about 30 days.

[0639] The method according to any one of Clauses 620 - 629, wherein the residual water content of the microspheres is between about 1% by mass and about 10% by mass.

[0640] Clause 631. The method according to any one of Clauses 620 - 630, wherein the dehydration comprises lyophilizing the microspheres or drying the microspheres using supercritical CO2.

[0641] Clause 632. The method according to any one of Clauses 620 - 631, wherein the sterilization comprises irradiating the microspheres with γ - radiation of 6 - 10 kGy.

[0642] Clause 633. The method according to any one of Clauses 620 - 632, wherein the precursor solution and / or the gelling bath further comprises an anti - inflammatory agent, a chemotherapeutic agent, an antioxidant, or a combination thereof.

[0643] Clause 634. The method according to Clause 633, wherein the microspheres comprise one or more of the following:

[0644] An anti - inflammatory agent selected from hyaluronic acid, ibuprofen, flurbiprofen, meloxicam, nabumetone, etodolac, ketorolac, ketoprofen, diflunisal, naproxen, diclofenac, celecoxib, mefenamic acid, etoricoxib, indomethacin, aspirin, arnica, curcumin, bromelain, and paracetamol;

[0645] An antioxidant selected from glutathione, α - tocopherol, ergothioneine, N - acetylcysteine, ascorbic acid, vitamin A, vitamin E, allopurinol, melatonin, resveratrol, bucillamine, turmeric, lycopene, lipoic acid, cobalamin, flavonoids, quercetin, ebselen, and edaravone; or

[0646] A corticosteroid selected from methylprednisolone, dexamethasone, triamcinolone acetonide, betamethasone, beclomethasone, and hydrocortisone.

[0647] Clause 635. A method for preparing a photopolymerizable microsphere capable of self - degrading upon rehydration, the method comprising:

[0648] Forming droplets from a precursor solution, the precursor solution comprising:

[0649] (i) An enzyme pretreated by varying temperature, by varying pH, and / or with a metal ion enzyme inhibitor;

[0650] (ii) A biomaterial comprising a photocrosslinkable moiety;

[0651] (iii) A photoinitiator;

[0652] Irradiating the droplets so that the biomaterial crosslinks to form a photopolymerized bio - derived microsphere encapsulating the enzyme; and

[0653] Dehydrate the microspheres and optionally sterilize them to substantially remove water from the microspheres.

[0654] Clause 636. The method according to clause 635, wherein the photocrosslinkable moiety is selected from acrylate group, methacrylate group, vinyl group and allyl group.

[0655] Clause 637. The method according to clause 635 or 636, wherein the precursor solution further comprises one or more cryoprotectants.

[0656] Clause 638. The method according to any one of clauses 635-637, wherein the cryoprotectant is selected from hydroxypropyl-β-cyclodextrin, trehalose, polyvinylpyrrolidone and dextran.

[0657] Clause 639. The method according to any one of clauses 635-638, wherein the enzyme is an enzyme that acts on a biomaterial and degrades the biomaterial.

[0658] Clause 640. The method according to any one of clauses 635-639, wherein the biomaterial comprises a polysaccharide, a protein or a glycoprotein.

[0659] Clause 641. The method according to any one of clauses 635-640, wherein the microspheres comprise alginate particles encapsulating alginate lyase, pectin particles encapsulating pectinase, hyaluronic acid particles encapsulating hyaluronidase, gelatin particles encapsulating matrix metalloproteinase or protease, albumin particles encapsulating peptidase, collagen particles encapsulating protease, fibrinogen particles encapsulating plasmin, fibroin particles encapsulating protease, starch particles encapsulating amylase, chitosan particles encapsulating chitosanase or lysozyme, agar / agarose particles encapsulating agarase, carrageenan particles encapsulating carrageenase, pullulan particles encapsulating pullulanase, dextran particles encapsulating dextranase, b-glycan particles encapsulating b-glycanase, cellulose particles encapsulating cellulase or lignin particles encapsulating ligninase.

[0660] Clause 642. The method according to any one of clauses 635-641, wherein the self-degradation of the microspheres is controlled by one or more of the following: pretreatment of the enzyme, concentration of the enzyme in the microspheres, enzyme activity, predetermined molecular weight of the biomaterial, and amount of time the droplets are irradiated.

[0661] Clause 643. The method according to any one of clauses 635-642, wherein the microspheres self-degrade in less than about 4 hours, in a time period greater than about 2 days to less than about 5 days, in a time period greater than about 5 days to less than about 30 days, or in greater than about 30 days.

[0662] The method according to any one of clauses 635 - 643, wherein:

[0663] The enzyme activity is between about 0.0075 U / mg and 0.25 U / mg of biomaterial, and the microspheres degrade within a time period greater than about 20 minutes to less than about 4 hours;

[0664] The enzyme activity is between about 0.005 U / mg and about ≥0.0025 U / mg of biomaterial, and the microspheres degrade within a time period greater than about 5 days to less than about 30 days; or

[0665] The enzyme activity is less than about 0.0025 U / mg of biomaterial, and the microspheres degrade within a time period greater than about 30 days.

[0666] Clause 645. The method according to any one of clauses 635 - 644, wherein the residual water content of the microspheres is between about 1% by mass and about 10% by mass.

[0667] Clause 646. The method according to any one of clauses 635 - 645, wherein dehydration includes freeze - drying the microspheres or drying the microspheres using supercritical CO2.

[0668] Clause 647. The method according to any one of clauses 635 - 646, wherein the sterilization includes irradiating the microspheres with γ - radiation at 6 - 10 kGy.

[0669] Clause 648. The method according to any one of clauses 635 - 647, wherein the precursor solution further comprises an anti - inflammatory agent, a chemotherapeutic agent, an antioxidant, or a combination thereof.

[0670] Clause 649. The method according to clause 648, wherein the microspheres comprise one or more of the following:

[0671] An anti - inflammatory agent selected from hyaluronic acid, ibuprofen, flurbiprofen, meloxicam, nabumetone, etodolac, ketorolac, ketoprofen, diflunisal, naproxen, diclofenac, celecoxib, mefenamic acid, etoricoxib, indomethacin, aspirin, arnica, curcumin, bromelain, and paracetamol,

[0672] An antioxidant selected from glutathione, α - tocopherol, ergothioneine, N - acetylcysteine, ascorbic acid, vitamin A, vitamin E, allopurinol, melatonin, resveratrol, bucillamine, turmeric, lycopene, lipoic acid, cobalamin, flavonoids, quercetin, ebselen, and edaravone; or

[0673] A corticosteroid selected from methylprednisolone, dexamethasone, triamcinolone acetonide, betamethasone, beclomethasone, and hydrocortisone.

[0674] Article 650. A method for preparing microspheres capable of self-degrading upon rehydration, the method comprising:

[0675] forming droplets from a precursor solution, the precursor solution comprising:

[0676] (i) a biomaterial comprising covalently crosslinkable moieties; and

[0677] (ii) a homobifunctional crosslinker or a heterobifunctional crosslinker;

[0678] covalently crosslinking the biomaterial to form bio-derived microspheres;

[0679] swelling an enzyme, which has been pretreated by varying temperature, by varying pH, and / or with a metal ion enzyme inhibitor, into the microspheres such that the bio-derived microspheres encapsulate the enzyme; and

[0680] dehydrating the microspheres and optionally sterilizing them, thereby substantially removing water from the microspheres.

[0681] Article 651. The method according to Article 650, wherein the covalently crosslinkable moieties comprise amino groups or carboxyl groups.

[0682] Article 652. The method according to Article 650 or 651, wherein the precursor solution further comprises one or more cryoprotectants.

[0683] Article 653. The method according to Article 652, wherein the cryoprotectant is selected from hydroxypropyl-β-cyclodextrin, trehalose, polyvinylpyrrolidone, and dextran.

[0684] Article 654. The method according to any one of Articles 650-653, wherein the enzyme is an enzyme that acts on the biomaterial and degrades the biomaterial.

[0685] Article 655. The method according to any one of Articles 650-654, wherein the biomaterial comprises a polysaccharide, a protein, or a glycoprotein.

[0686] Article 656. The method according to any one of Articles 650-655, wherein the microspheres comprise alginate particles encapsulating alginate lyase, pectin particles encapsulating pectinase, hyaluronic acid particles encapsulating hyaluronidase, gelatin particles encapsulating matrix metalloproteinase or protease, albumin particles encapsulating peptidase, collagen particles encapsulating protease, fibrinogen particles encapsulating plasmin, silk fibroin particles encapsulating protease, starch particles encapsulating amylase, chitosan particles encapsulating chitosanase or lysozyme, agar / agarose particles encapsulating agarase, carrageenan particles encapsulating carrageenase, pullulan particles encapsulating pullulanase, dextran particles encapsulating dextranase, β-glycan particles encapsulating β-glycanase, cellulose particles encapsulating cellulase, or lignin particles encapsulating ligninase.

[0687] Clause 657. The method according to any one of Clauses 650 - 656, wherein the self - degradation of the microspheres is controlled by one or more of the following: pretreatment of the enzyme, the concentration of the enzyme in the microspheres, the enzyme activity, the predetermined molecular weight of the biomaterial, and the homobifunctional cross - linker or heterobifunctional cross - linker used to cross - link the biomaterial.

[0688] Clause 658. The method according to any one of Clauses 650 - 657, wherein the microspheres self - degrade in less than about 4 hours, in a time period greater than about 2 days to less than about 5 days, in a time period greater than about 5 days to less than about 30 days, or in a time period greater than about 30 days.

[0689] Clause 659. The method according to any one of Clauses 650 - 658, wherein:

[0690] The enzyme activity is between about 0.0075 U / mg and 0.25 U / mg of biomaterial, and the microspheres degrade in a time period greater than about 20 minutes to less than about 4 hours;

[0691] The enzyme activity is between about 0.005 U / mg and about ≥0.0025 U / mg of biomaterial, and the microspheres degrade in a time period greater than about 5 days to less than about 30 days; or

[0692] The enzyme activity is less than about 0.0025 U / mg of biomaterial, and the microspheres degrade in a time period greater than about 30 days.

[0693] Clause 660. The method according to any one of Clauses 650 - 659, wherein the residual water content of the microspheres is between about 1% by mass and about 10% by mass.

[0694] Clause 661. The method according to any one of Clauses 650 - 660, wherein dehydration includes freeze - drying the microspheres or drying the microspheres using supercritical CO2.

[0695] Clause 662. The method according to any one of Clauses 650 - 661, wherein the sterilization includes irradiating the microspheres with γ - radiation at 6 - 10 kGy.

[0696] Clause 663. The method according to any one of Clauses 650 - 662, wherein the precursor solution further comprises an anti - inflammatory agent, a chemotherapeutic agent, an antioxidant, or a combination thereof.

[0697] Clause 664. The method according to Clause 663, wherein the microspheres comprise one or more of the following:

[0698] An anti-inflammatory agent selected from hyaluronic acid, ibuprofen, flurbiprofen, meloxicam, nabumetone, etodolac, ketorolac, ketoprofen, diflunisal, naproxen, diclofenac, celecoxib, mefenamic acid, etoricoxib, indomethacin, aspirin, arnica, curcumin, bromelain, and acetaminophen,

[0699] An antioxidant selected from glutathione, α-tocopherol, ergothioneine, N-acetylcysteine, ascorbic acid, vitamin A, vitamin E, allopurinol, melatonin, resveratrol, bucillamine, turmeric, lycopene, lipoic acid, cobalamin, flavonoids, quercetin, ebselen, and edaravone; or

[0700] A corticosteroid selected from methylprednisolone, dexamethasone, triamcinolone, betamethasone, beclomethasone, and hydrocortisone.

[0701] Clause 665. A method for preparing microspheres capable of self-degrading upon rehydration, the method comprising:

[0702] Forming droplets from a precursor solution, the precursor solution comprising:

[0703] (i) A biomaterial comprising a covalently crosslinkable moiety;

[0704] (ii) An enzyme that has been pretreated by varying temperature, by varying pH, and / or with a metal ion enzyme inhibitor; and

[0705] (iii) A homobifunctional crosslinker or a heterobifunctional crosslinker;

[0706] Covalently crosslinking the biomaterial to form a bio-derived microsphere encapsulating the enzyme; and

[0707] Dehydrating the microspheres and optionally sterilizing them to substantially remove water from the microspheres.

[0708] Clause 666. The method according to Clause 665, wherein the covalently crosslinkable moiety comprises an amino group or a carboxyl group.

[0709] Clause 667. The method according to Clause 665 or 666, wherein the precursor solution further comprises one or more cryoprotectants.

[0710] Clause 668. The method according to Clause 667, wherein the cryoprotectant is selected from hydroxypropyl-β-cyclodextrin, trehalose, polyvinylpyrrolidone, and dextran.

[0711] Clause 669. The method according to any one of Clauses 665-668, wherein the enzyme is an enzyme that acts on the biomaterial and degrades the biomaterial.

[0712] Method according to any one of clauses 665 - 669, wherein the biological material comprises a polysaccharide, a protein or a glycoprotein.

[0713] Clause 671. Method according to any one of clauses 665 - 670, wherein the microspheres comprise alginate particles encapsulating alginate lyase, pectin particles encapsulating pectinase, hyaluronic acid particles encapsulating hyaluronidase, gelatin particles encapsulating matrix metalloproteinase or protease, albumin particles encapsulating peptidase, collagen particles encapsulating protease, fibrinogen particles encapsulating plasmin, silk fibroin particles encapsulating protease, starch particles encapsulating amylase, chitosan particles encapsulating chitosanase or lysozyme, agar / agarose particles encapsulating agarase, carrageenan particles encapsulating carrageenase, pullulan particles encapsulating pullulanase, dextran particles encapsulating dextranase, β-glycan particles encapsulating β-glycanase, cellulose particles encapsulating cellulase or lignin particles encapsulating ligninase.

[0714] Clause 672. Method according to any one of clauses 665 - 671, wherein the self-degradation of the microspheres is controlled by one or more of: pretreatment of the enzyme, the concentration of the enzyme in the microspheres, the enzyme activity, the predetermined molecular weight of the biological material, and the homobifunctional crosslinking agent or heterobifunctional crosslinking agent used to crosslink the biological material.

[0715] Clause 673. Method according to any one of clauses 665 - 672, wherein the microspheres self-degrade in less than about 4 hours, in a time period greater than about 2 days to less than about 5 days, in a time period greater than about 5 days to less than about 30 days, or in a time period greater than about 30 days.

[0716] Clause 674. Method according to any one of clauses 665 - 673, wherein:

[0717] The enzyme activity is between about 0.0075 U / mg and 0.25 U / mg of biological material, and the microspheres degrade in a time period greater than about 20 minutes to less than about 4 hours;

[0718] The enzyme activity is between about 0.005 U / mg and about ≥0.0025 U / mg of biological material, and the microspheres degrade in a time period greater than about 5 days to less than about 30 days; or

[0719] The enzyme activity is less than about 0.0025 U / mg of biological material, and the microspheres degrade in a time period greater than about 30 days.

[0720] Clause 675. Method according to any one of clauses 665 - 674, wherein the residual water content of the microspheres is between about 1% by mass and about 10% by mass.

[0721] Article 676. The method according to any one of Articles 665 - 675, wherein the dehydration comprises lyophilizing the microspheres or drying the microspheres using supercritical CO2.

[0722] Article 677. The method according to any one of Articles 665 - 676, wherein the sterilization comprises irradiating the microspheres with γ-radiation of 6 - 10 kGy.

[0723] Article 678. The method according to any one of Articles 665 - 677, wherein the precursor solution further comprises an anti-inflammatory agent, a chemotherapeutic agent, an antioxidant, or a combination thereof.

[0724] Article 679. The method according to Article 678, wherein the microspheres comprise one or more of the following:

[0725] An anti-inflammatory agent selected from hyaluronic acid, ibuprofen, flurbiprofen, meloxicam, nabumetone, etodolac, ketorolac, ketoprofen, diflunisal, naproxen, diclofenac, celecoxib, mefenamic acid, etoricoxib, indomethacin, aspirin, arnica, curcumin, bromelain, and paracetamol,

[0726] An antioxidant selected from glutathione, α-tocopherol, ergothioneine, N-acetylcysteine, ascorbic acid, vitamin A, vitamin E, allopurinol, melatonin, resveratrol, bucillamine, turmeric, lycopene, lipoic acid, cobalamin, flavonoids, quercetin, ebselen, and edaravone; or

[0727] A corticosteroid selected from methylprednisolone, dexamethasone, triamcinolone acetonide, betamethasone, beclomethasone, and hydrocortisone.

[0728] Article 680. A method for preparing thermogel microspheres capable of self-degrading upon rehydration, the method comprising:

[0729] Heating a precursor solution containing a biomaterial to melt the biomaterial;

[0730] Adding an enzyme pretreated by a changing temperature, by a changing pH, and / or with a metal ion enzyme inhibitor to the precursor solution;

[0731] Forming droplets from the precursor solution;

[0732] Cooling the droplets to form thermogel bioderived microspheres encapsulating the enzyme; and

[0733] Dehydrating the microspheres and optionally sterilizing them to substantially remove water from the microspheres.

[0734] Article 681. The method according to Article 680, wherein the precursor solution further comprises one or more cryoprotectants.

[0735] Clause 682. The method according to Clause 681, wherein the cryoprotectant is selected from hydroxypropyl-β-cyclodextrin, trehalose, polyvinylpyrrolidone, and dextran.

[0736] Clause 683. The method according to any one of Clauses 680-682, wherein the enzyme is an enzyme that acts on a biomaterial and degrades the biomaterial.

[0737] Clause 684. The method according to any one of Clauses 680-683, wherein the biomaterial comprises a polysaccharide, a protein, or a glycoprotein.

[0738] Clause 685. The method according to any one of Clauses 680-684, wherein the microspheres comprise pectin particles encapsulating pectinase, gelatin particles encapsulating matrix metalloproteinase or protease, albumin particles encapsulating peptidase, collagen particles encapsulating protease, fibrinogen particles encapsulating plasmin, silk fibroin particles encapsulating protease, starch particles encapsulating amylase, chitosan particles encapsulating chitosanase or lysozyme, or agar / agarose particles encapsulating agarase.

[0739] Clause 686. The method according to any one of Clauses 680-685, wherein the self-degradation of the microspheres is controlled by one or more of the following: pretreatment of the enzyme, the concentration of the enzyme in the microspheres, the enzyme activity, and the predetermined molecular weight of the biomaterial.

[0740] Clause 687. The method according to any one of Clauses 680-686, wherein the microspheres self-degrade in less than about 4 hours, in a time period greater than about 2 days to less than about 5 days, in a time period greater than about 5 days to less than about 30 days, or in greater than about 30 days.

[0741] Clause 688. The method according to any one of Clauses 680-687, wherein:

[0742] the enzyme activity is between about 0.0075 U / mg and 0.25 U / mg of the biomaterial, and the microspheres degrade in a time period greater than about 20 minutes to less than about 4 hours;

[0743] the enzyme activity is between about 0.005 U / mg and about ≥0.0025 U / mg of the biomaterial, and the microspheres degrade in a time period greater than about 5 days to less than about 30 days; or

[0744] the enzyme activity is less than about 0.0025 U / mg of the biomaterial, and the microspheres degrade in a time period greater than about 30 days.

[0745] Clause 689. The method according to any one of Clauses 680-688, wherein the residual water content of the microspheres is between about 1% by mass and about 10% by mass.

[0746] Clause 690. The method according to any one of Clauses 680 - 689, wherein the dehydration comprises freeze-drying the microspheres or drying the microspheres using supercritical CO2.

[0747] Clause 691. The method according to any one of Clauses 680 - 690, wherein the sterilization comprises irradiating the microspheres with γ-radiation of 6 - 10 kGy.

[0748] Clause 692. The method according to any one of Clauses 680 - 691, wherein the precursor solution further comprises an anti-inflammatory agent, a chemotherapeutic agent, an antioxidant, or a combination thereof.

[0749] Clause 693. The method according to Clause 692, wherein the microspheres comprise one or more of the following:

[0750] An anti-inflammatory agent selected from hyaluronic acid, ibuprofen, flurbiprofen, meloxicam, nabumetone, etodolac, ketorolac, ketoprofen, diflunisal, naproxen, diclofenac, celecoxib, mefenamic acid, etoricoxib, indomethacin, aspirin, arnica, curcumin, bromelain, and paracetamol,

[0751] An antioxidant selected from glutathione, α-tocopherol, ergothioneine, N-acetylcysteine, ascorbic acid, vitamin A, vitamin E, allopurinol, melatonin, resveratrol, bucillamine, turmeric, lycopene, dihydrolipoic acid, cobalamin, flavonoids, quercetin, ebselen, and edaravone; or

[0752] A corticosteroid selected from methylprednisolone, dexamethasone, triamcinolone acetonide, betamethasone, beclomethasone, and hydrocortisone.

[0753] Clause 694. A method for preparing self-degradable microspheres, the method comprising:

[0754] Forming a precursor solution comprising:

[0755] (i) an enzyme; and

[0756] (ii) a biomaterial;

[0757] Passing the precursor solution through a needle under the influence of an electrostatic potential to form droplets; and

[0758] Contacting the droplets with a gelling bath comprising divalent metal ions, thereby crosslinking the biomaterial to form bio-derived microspheres encapsulating the enzyme.

[0759] Clause 695. The method according to Clause 694, further comprising dehydrating the microspheres and optionally sterilizing them, thereby substantially removing water from the microspheres to form microspheres that are capable of self-degrading upon rehydration.

[0760] Article 696. The method according to Article 694 or 695, wherein the precursor solution further comprises one or more cryoprotectants.

[0761] Article 697. The method according to Article 696, wherein the cryoprotectant is selected from hydroxypropyl-β-cyclodextrin, trehalose, polyvinylpyrrolidone, and dextran.

[0762] Article 698. The method according to any one of Articles 694-697, wherein the electrostatic potential is between about 1 kV and about 5 kV.

[0763] Article 699. The method according to any one of Articles 694-698, wherein the enzyme is an enzyme that acts on a biomaterial and degrades the biomaterial.

[0764] Article 700. The method according to any one of Articles 694-699, wherein the biomaterial comprises a polysaccharide, a protein, or a glycoprotein.

[0765] Article 701. The method according to any one of Articles 694-700, wherein the microspheres comprise alginate particles encapsulating alginate lyase, pectin particles encapsulating pectinase, hyaluronic acid particles encapsulating hyaluronidase, gelatin particles encapsulating matrix metalloproteinase or protease, albumin particles encapsulating peptidase, collagen particles encapsulating protease, fibrinogen particles encapsulating plasmin, silk fibroin particles encapsulating protease, starch particles encapsulating amylase, chitosan particles encapsulating chitosanase or lysozyme, agar / agarose particles encapsulating agarase, carrageenan particles encapsulating carrageenase, pullulan particles encapsulating pullulanase, dextran particles encapsulating dextranase, b-glycan particles encapsulating b-glycanase, cellulose particles encapsulating cellulase, or lignin particles encapsulating ligninase.

[0766] Article 702. The method according to any one of Articles 694-701, wherein the self-degradation of the microspheres is controlled by one or more of the following: pretreatment of the enzyme, concentration of the enzyme in the microspheres, enzyme activity, predetermined molecular weight of the biomaterial, divalent metal ions used for crosslinking the biomaterial, and amount of divalent metal ions used for crosslinking the biomaterial.

[0767] Article 703. The method according to any one of Articles 694-702, wherein the microspheres self-degrade in less than about 4 hours, in a time period greater than about 2 days to less than about 5 days, in a time period greater than about 5 days to less than about 30 days, or in a time period greater than about 30 days.

[0768] Article 704. The method according to any one of Articles 694-703, wherein:

[0769] The enzyme activity is between about 0.0075 U / mg and 0.25 U / mg of biomaterial, and the microspheres degrade within a time period greater than about 20 minutes to less than about 4 hours;

[0770] The enzyme activity is between about 0.005 U / mg and about ≥0.0025 U / mg of biomaterial, and the microspheres degrade within a time period greater than about 5 days to less than about 30 days; or

[0771] The enzyme activity is less than about 0.0025 U / mg of biomaterial, and the microspheres degrade within a time period greater than about 30 days.

[0772] Clause 705. The method according to any one of Clauses 694 - 704, wherein the residual water content of the microspheres is between about 1% by mass and about 10% by mass.

[0773] Clause 706. The method according to any one of Clauses 695 - 705, wherein dehydration includes freeze - drying the microspheres or drying the microspheres using supercritical CO2.

[0774] Clause 707. The method according to any one of Clauses 695 - 706, wherein the sterilization includes irradiating the microspheres with γ - radiation of 6 - 10 kGy.

[0775] Clause 708. The method according to any one of Clauses 694 - 707, wherein the precursor solution further comprises an anti - inflammatory agent, a chemotherapeutic agent, an antioxidant, or a combination thereof.

[0776] Clause 709. The method according to Clause 708, wherein the microspheres comprise one or more of the following:

[0777] An anti - inflammatory agent selected from hyaluronic acid, ibuprofen, flurbiprofen, meloxicam, nabumetone, etodolac, ketorolac, ketoprofen, diflunisal, naproxen, diclofenac, celecoxib, mefenamic acid, etoricoxib, indomethacin, aspirin, arnica, curcumin, bromelain, and paracetamol,

[0778] An antioxidant selected from glutathione, α - tocopherol, ergothioneine, N - acetylcysteine, ascorbic acid, vitamin A, vitamin E, allopurinol, melatonin, resveratrol, bucillamine, turmeric, lycopene, dihydrolipoic acid, cobalamin, flavonoids, quercetin, ebselen, and edaravone; or

[0779] A corticosteroid selected from methylprednisolone, dexamethasone, triamcinolone acetonide, betamethasone, beclomethasone, and hydrocortisone.

[0780] Article 694. A method of inducing self-degrading embolization in a subject in need thereof, comprising administering a plurality of microspheres as described in any one of Articles 601-619 into a blood vessel of the subject.

[0781] Article 695. The method according to Article 694, wherein the blood vessel is the geniculate artery.

[0782] Article 696. The method according to Article 694 or 695, wherein the method induces prostatic artery embolization, induces uterine artery embolization, or the microspheres comprise a chemotherapeutic agent or are mixed with a chemotherapeutic agent, and the method induces transarterial chemoembolization (TACE).

[0783] Article 697. A method of treating a disease or disorder in a subject in need thereof, comprising administering a plurality of microspheres as described in any one of Articles 601-619 to the subject.

[0784] Article 698. The method according to Article 697, wherein the disease or disorder is tendinopathy.

[0785] Article 699. The method according to Article 697, wherein the disease or disorder is selected from osteoarthritis, frozen shoulder, tennis elbow (lateral epicondylitis), golfer's elbow (medial epicondylopathy), pitcher's elbow (flexor tendinitis), Achilles tendinopathy, plantar fasciitis, symptomatic accessory navicular pain, hamstring tendinopathy, jumper's knee (patellar tendinitis), runner's knee (patellofemoral pain syndrome (PFPS)), pes anserine bursitis (knee pain), posterior tibial tendon disease, wrist (TFCC - triangular fibrocartilage complex) tendon disease, trigger finger (stenosing flexor tenosynovitis), and hemarthrosis.

[0786] Article 700. A method of rapidly degrading microspheres in a subject, comprising administering an emergency rescue solution to the subject, wherein a plurality of microspheres as described in any one of Articles 601-619 have been previously administered to the subject, and the emergency rescue solution comprises an enzyme capable of degrading the microspheres.

[0787] Article 701. The method according to Article 700, wherein the enzyme is complementary to the biomaterial used to form the plurality of microspheres.

[0788] Article 702. The method according to Article 700 or 701, wherein:

[0789] the microspheres comprise alginate particles encapsulating alginate lyase and the enzyme is alginate lyase,

[0790] the microspheres comprise pectin particles encapsulating pectinase and the enzyme is pectinase,

[0791] the microspheres comprise hyaluronic acid particles encapsulating hyaluronidase and the enzyme is hyaluronidase,

[0792] The microspheres comprise gelatin particles encapsulating a matrix metalloproteinase or a protease, and the enzyme is a matrix metalloproteinase or a protease.

[0793] The microspheres comprise albumin particles encapsulating a peptidase, and the enzyme is a peptidase.

[0794] The microspheres comprise collagen particles encapsulating a protease, and the enzyme is a protease.

[0795] The microspheres comprise fibrinogen particles encapsulating plasmin, and the enzyme is plasmin.

[0796] The microspheres comprise fibroin particles encapsulating a protease, and the enzyme is a protease.

[0797] The microspheres comprise starch particles encapsulating amylase, and the enzyme is amylase.

[0798] The microspheres comprise chitosan particles encapsulating chitosanase or lysozyme, and the enzyme is chitosanase or lysozyme.

[0799] The microspheres comprise agar / agarose particles encapsulating agarase, and the enzyme is agarase.

[0800] The microspheres comprise carrageenan particles encapsulating carrageenase, and the enzyme is carrageenase.

[0801] The microspheres comprise pullulan particles encapsulating pullulanase, and the enzyme is pullulanase.

[0802] The microspheres comprise dextran particles encapsulating dextranase, and the enzyme is dextranase.

[0803] The microspheres comprise β-glycan particles encapsulating β-glycanase, and the enzyme is β-glycanase.

[0804] The microspheres comprise cellulose particles encapsulating cellulase, and the enzyme is cellulase, or

[0805] The microspheres comprise lignin particles encapsulating ligninase, and the enzyme is ligninase.

[0806] Clause 703. The method according to any one of Clauses 700 - 702, wherein the emergency rescue solution further comprises a divalent metal chelator.

[0807] Clause 704. A method for rapidly degrading divalent metal ion-crosslinked microspheres in a subject, comprising administering an emergency rescue solution to the subject, wherein the divalent metal ion-crosslinked microspheres according to any one of Clauses 601 - 619 have been previously administered to the subject, and the emergency rescue solution comprises an anion, a phosphate buffer, or a combination thereof.

[0808] Clause 705. The method according to Clause 704, wherein the anion comprises citrate.

[0809] Clause 706. The method according to Clause 704, wherein the phosphate buffer comprises phosphate buffered saline.

[0810] Clause 707. The method according to any one of Clauses 704-706, wherein the microspheres comprise alginate particles encapsulating alginate lyase, pectin particles encapsulating pectinase, or carrageenan particles encapsulating carrageenase.

[0811] Clause 708. The method according to Clause 704 or 705, wherein the microspheres comprise alginate particles encapsulating alginate lyase, pectin particles encapsulating pectinase, or carrageenan particles encapsulating carrageenase; and wherein the anion comprises citrate.

[0812] Clause 709. The method according to Clause 704 or 706, wherein the microspheres comprise alginate particles encapsulating alginate lyase, pectin particles encapsulating pectinase, or carrageenan particles encapsulating carrageenase; and wherein the phosphate buffer comprises phosphate buffered saline.

[0813] Clause 710. A kit comprising:

[0814] A plurality of microspheres according to any one of Clauses 601-619; and

[0815] An enzyme that can rapidly degrade the microspheres when dissolved to form a solution.

[0816] Clause 711. The kit according to Clause 710, wherein the enzyme is complementary to the biomaterial used to form the plurality of microspheres.

[0817] Clause 712. The kit according to Clause 710 or 711, wherein:

[0818] The microspheres comprise alginate particles encapsulating alginate lyase and the enzyme is alginate lyase,

[0819] The microspheres comprise pectin particles encapsulating pectinase and the enzyme is pectinase,

[0820] The microspheres comprise hyaluronic acid particles encapsulating hyaluronidase and the enzyme is hyaluronidase,

[0821] The microspheres comprise gelatin particles encapsulating matrix metalloproteinase or protease and the enzyme is matrix metalloproteinase or protease,

[0822] The microspheres comprise albumin particles encapsulating peptidase and the enzyme is peptidase,

[0823] The microspheres contain collagen particles encapsulating a protease and the enzyme is a protease,

[0824] The microspheres contain fibrinogen particles encapsulating plasmin and the enzyme is plasmin,

[0825] The microspheres contain fibroin particles encapsulating a protease and the enzyme is a protease,

[0826] The microspheres contain starch particles encapsulating amylase and the enzyme is amylase,

[0827] The microspheres contain chitosan particles encapsulating chitosanase or lysozyme and the enzyme is chitosanase or lysozyme,

[0828] The microspheres contain agar / agarose particles encapsulating agarase and the enzyme is agarase,

[0829] The microspheres contain carrageenan particles encapsulating carrageenase and the enzyme is carrageenase,

[0830] The microspheres contain pullulan particles encapsulating pullulanase and the enzyme is pullulanase,

[0831] The microspheres contain dextran particles encapsulating dextranase and the enzyme is dextranase,

[0832] The microspheres contain β-glycan particles encapsulating β-glycanase and the enzyme is β-glycanase,

[0833] The microspheres contain cellulose particles encapsulating cellulase and the enzyme is cellulase, or

[0834] The microspheres contain lignin particles encapsulating ligninase and the enzyme is ligninase.

[0835] Clause 713. A kit comprising:

[0836] A plurality of divalent metal ion crosslinked microspheres according to any one of Clauses 601 - 619; and

[0837] An inorganic salt that can rapidly degrade the microspheres when dissolved to form a solution.

[0838] Clause 714. The kit according to Clause 713, wherein the microspheres contain alginate particles encapsulating alginate lyase, pectin particles encapsulating pectinase, or carrageenan particles encapsulating carrageenase.

[0839] Clause 715. The kit according to Clause 713 or 714, wherein the inorganic salt releases citrate or phosphate when dissolved to form a solution.

[0840] Example

[0841] Example 1: Alginate Lyase Concentration - Dependent Degradation of Alginate Particles

[0842] Schematic diagrams of the preparation of alginate particles are shown in Figure 2A - 2C and Figure 3A - 3C . Sodium alginate with a certain viscosity (5 - 40 cP, conditions 1% w / v in water, at 25 °C) was dissolved in deionized water to prepare a stock solution with a concentration of 4% w / v. Similarly, a stock solution of alginate lyase with a concentration of 50 U / mL was prepared by dissolving 5 mg of enzyme powder (equivalent to 50 U) in 1 mL of deionized water. To prepare an alginate lyase - sodium alginate precursor solution with a final concentration of 5 U / mL (0.25 U / mg alginate) or 0.5 U / ml (0.025 U / mg alginate) alginate lyase and 2% w / v sodium alginate, 0.1 mL or 0.01 mL of alginate lyase was mixed with 0.5 ml of 4% w / v sodium alginate for 30 seconds, and the volume was made up to 1 mL with deionized water.

[0843] Under continuous stirring for 5 minutes, the precursor alginate lyase - alginate solution was added dropwise to a gelling bath containing 10% w / v calcium chloride to achieve calcium 2+ crosslinked alginate particles loaded with alginate lyase. Then, the particles were sieved or centrifuged and washed three times with deionized water for 1 minute each to remove excess calcium chloride. The washed calcium 2+ crosslinked alginate particles loaded with alginate lyase were dispersed in 10 mM phosphate buffer at pH 6.8 and incubated at 37 °C for the desired duration to evaluate the degradation of alginate particles. The degradation of calcium ion - crosslinked alginate particles loaded with 5 units (U) (0.25 U / mg alginate) and 0.5 U (0.05 U / mg sodium alginate) alginate lyase is shown in Figure 4A . Alginate particles loaded with 5 U of enzyme degraded rapidly within 12 hours, while particles loaded with 0.5 U of enzyme degraded at a slower rate and could not reach the absorbance level similar to that of 5 U - loaded alginate particles after 36 hours. From Figure 4B , alginate particles loaded with 5 U of alginate lyase were completely degraded within 12 hours, while samples of alginate particles loaded with 0.5 U of alginate lyase showed partially degraded particles within 36 hours. In the control sample (without enzyme), calcium ion - complexed alginate particles remained intact. These results demonstrate the concentration - dependent degradation of alginate particles by alginate lyase.

[0844] Example 2: Alginate Lyase Concentration - Dependent Degradation of Alginate Particles Prepared from High - Viscosity Alginate

[0845] Sodium alginate with high viscosity (viscosity 144 cP, conditions 1% w / v in water, at 25 °C) was dissolved in deionized water to prepare a stock solution with a concentration of 3% w / v. Similarly, a stock solution of alginate lyase with a concentration of 50 U / ml was prepared by dissolving 5 mg of enzyme powder (equivalent to 50 U) in 1 ml of deionized water. To prepare alginate lyase-sodium alginate precursor solutions with final concentrations of 1 U / ml (or 0.05 U / mg alginate), 0.5 U / ml (or 0.025 U / mg alginate), and 0.25 U / ml (or 0.0125 U / mg alginate) alginate lyase and 2% w / v sodium alginate, 0.02 ml, 0.01 ml, 0.005 ml of alginate lyase was mixed with 0.5 ml of 3% w / v sodium alginate for 30 seconds, and the volume was made up to 1 ml with deionized water.

[0846] Under continuous stirring for 5 minutes, the precursor alginate lyase-sodium alginate solution was added dropwise to a gelling bath containing 2% w / v calcium chloride to achieve calcium 2+ complexed alginate particles loaded with alginate lyase. Then, the particles were separated by sieving or centrifugation and washed three times with deionized water for 1 minute each to remove excess calcium chloride. The washed calcium 2+ complexed alginate particles loaded with alginate lyase were dispersed in 10 mM phosphate buffer at pH 6.5 and incubated at 37 °C for the desired duration to evaluate the degradation of alginate particles. The degradation of calcium ion-complexed alginate particles loaded with 1 U, 0.5 U, and 0.25 U of alginate lyase was as Figure 5 shown. When compared with the particles loaded with 0.5 U and 0.25 U of enzyme, the alginate particles loaded with 1 U of enzyme degraded rapidly within 120 hours. In the control sample (without enzyme), the Ca 2+ crosslinked alginate particles remained intact. These results demonstrate the concentration-dependent degradation of alginate particles by alginate lyase.

[0847] Example 3: pH - Dependent Regulation of Alginate Lyase Conformation / Activity

[0848] To study the pH-dependent regulation of lyase conformation / activity, alginate lyase was exposed to different pHs and subjected to fluorescence spectroscopy. Generally, the open conformation of the enzyme inactivates or reduces the enzyme catalytic activity, while the further stabilization of the native structure improves the enzyme catalytic activity. From Figure 6Among them, when compared with the native enzyme at pH 7.0 (10 mM, phosphate buffer), the fluorescence of the native enzyme (1 U / ml or 0.05 U / mg alginate) at acidic pH 4.6 (acetate buffer) was at a lower level. The decrease in fluorescence at acidic pH indicates an open conformation of the enzyme. When the pH of the enzyme solution was changed to pH 4.6 - 7.0, a restored or enhanced fluorescence was found to be at a level similar to that of the native enzyme at pH 7.0. This demonstrates that the alginate lyase has a reversible conformation for reactivation of enzyme activity in response to changes in the solution pH. Therefore, by changing the pH of the alginate lyase-alginate precursor or gelling bath solution, initial degradation of the particles during the manufacture of divalent metal ion particles loaded with alginate lyase occurred. When reconstituting the alginate particles complexed with divalent metal ions loaded with alginate lyase in an aqueous solution at neutral pH (6.5 - 7.5), the activity of the alginate lyase can be restored to obtain customized degradation of the alginate particles.

[0849] Example 4: Effect of pH on the preparation of Ca 2+ crosslinked alginate microspheres loaded with alginate lyase

[0850] The alginate lyase and high-viscosity alginate (viscosity 144 cP, conditions 1% w / v in water, at 25 °C) were dissolved in 0.1 M sodium acetate buffer at pH 4, and the final concentrations were 5 U / ml and 1.5% w / v, respectively. Similarly, an alginate lyase-high-viscosity alginate (viscosity 144 cps, conditions 1% w / v in water, at 25 °C) solution was also prepared in 0.01 M phosphate buffer at pH 6.5, with final concentrations of 5 U / ml and 1.5% w / v, respectively. The two solutions were incubated at 4 °C for 15 minutes and then dropped into 2% w / v calcium chloride solution to obtain calcium ion-crosslinked alginate microspheres loaded with alginate lyase. The crosslinked microspheres were washed three times in deionized water, 1 minute each time. The microspheres prepared in acetate buffer showed a spherical shape ( Figure 7 (a)). On the other hand, the microspheres obtained from phosphate buffer were irregular in shape and partially degraded ( Figure 7 (b)). These results indicate that low pH reduces the catalytic activity of the enzyme and prevents the degradation of alginate, thus contributing to obtaining spherical microspheres. This method of preparing microspheres increases the processing window, which can contribute to improving the yield of alginate microspheres loaded with alginate lyase.

[0851] Example 5: From Alginate - Alginate Lyase Precursor Pretreated with Acidic pH (Acetate Buffer, pH 4) Ca prepared in solution 2+ Degradation of crosslinked alginate lyase-alginate microspheres

[0852] Alginate lyase and high-viscosity alginate (viscosity 144 cP, conditions 1% w / v in water, at 25 °C) were dissolved in 0.1 M sodium acetate buffer at pH 4, with final concentrations of 5 U / ml (0.25 U / mg alginate) and 1.5% w / v, respectively. Similarly, an alginate lyase-high-viscosity alginate (viscosity 144 cP, conditions 1% w / v in water, at 25 °C) solution was also prepared in 0.01 M phosphate buffer at pH 6.5, with final concentrations of 5 U / ml and 1.5% w / v, respectively. The two solutions were incubated at 4 °C for 15 minutes and then dropped into a 2% w / v calcium chloride solution to obtain calcium ion-crosslinked alginate microspheres loaded with alginate lyase. The crosslinked microspheres were washed three times in deionized water for 1 minute each. The microspheres prepared in acetate buffer showed spherical shapes ( Figure 8 (a)). On the other hand, the microspheres obtained from phosphate buffer were irregular in shape and partially degraded ( Figure 8 (b)). These results indicate that low pH reduces the catalytic activity of the enzyme and prevents the degradation of alginate, thus contributing to the formation of spherical microspheres. This method of preparing microspheres increases the processing window by minimizing the degradation of alginate by alginate lyase in the precursor solution, which can help improve the yield of alginate microspheres loaded with alginate lyase. The two microspheres were incubated in 0.01 M phosphate buffer and / or supplemented with 0.1 N NaOH to achieve pH 6.5 (in the case of the precursor solution pretreated with acetate buffer) for 72 hours. The acetate buffer-treated alginate-alginate lyase microspheres were completely degraded without visible signs of residue, as shown in Figure 8 (c). On the other hand, the phosphate buffer-treated alginate-alginate lyase microspheres showed some white residues, as observed in Figure 8 (d). The degradation of these particles was also determined using UV-visible spectroscopy, where the degradation products of alginate microspheres were determined by their absorbance at 235 nm. When compared with equivalent microspheres treated with phosphate buffer, it was observed that the acetate buffer-treated alginate lyase-alginate Ca 2+ crosslinked microspheres showed greater degradation ( Figure 8 (e)). These results indicate the pH-dependent reversible activity of alginate lyase, where by exposure to low pH, the enzyme is partially and reversibly inhibited or modulated, which enables the loading of the desired amount of enzyme in alginate microspheres without degrading the alginate matrix. The enzyme encapsulated in alginate microspheres is reversibly activated by exposure to the optimal pH, resulting in the degradation of alginate microspheres.

[0853] Example 6: Acidic pH - Dependent Reversible Activity of Alginate Lyase

[0854] The alginate lyase and high-viscosity alginate (viscosity 144 cP, conditions 1% w / v in water, at 25 °C) were dissolved in 0.1 M acetate buffer (pH 4.0) and 0.01 M phosphate buffer (pH 6.5), with final concentrations of 1 U / ml (0.05 U / mg alginate) and 0.1% w / v, respectively. The sample names for the corresponding reactions were alginate-AL A.B and alginate-AL P.B. The temperature of these solutions was maintained at 1 - 4 °C and 37 °C for 30 minutes. After incubation, the reaction was terminated by adding 0.1 N NaOH. Similarly, the alginate lyase was pre-incubated in acetate buffer for 15 minutes and then mixed with high-viscosity alginate dissolved in 0.01 M phosphate buffer supplemented with 0.1 N NaOH to achieve an optimal pH of 6.5, with final concentrations of 0.1 U / ml (0.005 U / mg alginate) and 1% w / v, respectively. The solution was incubated at 1 - 4 °C and 37 °C for 30 minutes. After incubation, the reaction was terminated by adding 0.1 N NaOH (sample name alginate(P.B)-AL(A.B)). After terminating the reaction, the enzyme activity of the alginate lyase was determined by the absorbance of the degradation product at a wavelength of 235 nm.

[0855] It was observed from Figure 9 that the enzyme activity of alginate-AL A.B decreased at 1 - 4 °C and 37 °C when compared with the alginate-AL P.B sample. Additionally, it was observed from the alginate-AL P.B sample that the effect of low temperature on reducing the alginate lyase activity was negligible. Furthermore, when compared with the alginate-AL P.B sample, the alginate lyase pretreated with acetate buffer mixed with alginate dissolved in phosphate buffer (alginate(P.B)-AL(A.B)) showed a 4-fold decrease in enzyme activity at 4 °C. This indicates that the acetate buffer partially reduces the enzyme activity at 1 - 4 °C. Significantly, the alginate lyase activity increased significantly at 37 °C, and its level was found to be similar to that of the alginate-AL P.B sample. This increase in activity shows pH-dependent reversible alginate lyase activity, where the enzyme is partially and reversibly inhibited or modulated by exposure to low pH and restored by changing to the optimal pH of the solution. These results were further supported by Figure 9 A-9C.

[0856] Example 7: Using Cryoprotectants PVP 40kDa (0.5%, w / v) and Trehalose (0.5%, w / v) to Load Ca of alginate lyase 2+ Freeze-drying of crosslinked alginate microspheres

[0857] Dissolve 1.5% w / v sodium alginate with high viscosity (viscosity 144 cP, condition 1% w / v in water, at 25 °C), PVP 40 kDa (0.5% w / v), and trehalose (0.5% w / v) in deionized water, and stir on a magnetic stirrer at 1 - 4 °C for half an hour / 45 minutes to obtain a homogeneous dispersion. Then, add 5 U of alginate lyase to the dispersion and mix for 1 minute. Dropwise add this solution into 2% w / v CaCl₂ containing PVP 40 kDa (0.5% w / v) and trehalose (0.5% w / v), and stir for 15 minutes to obtain Ca 2+ crosslinked alginate microspheres loaded with alginate lyase containing PVP 40 kDa and trehalose. Wash these microspheres further 3 times with deionized water, 1 minute each time, and expose them to liquid nitrogen for 30 seconds to 2 minutes. Use a freeze dryer set at -57 °C under ultra-high vacuum to lyophilize the frozen microspheres for 24 hours. From Figure 11 In A and 11A’, when compared with non-lyophilized microspheres, the freeze-dried or lyophilized microspheres showed no change in shape, thus indicating that the shape of the microspheres was maintained.

[0858] Example 8: Using Cryoprotectant Hydroxypropyl - β - Cyclodextrin (0.5%, w / v) to Load Alginate Lyase of Ca 2+ Lyophilized crosslinked alginate microspheres

[0859] Dissolve 1.5% w / v sodium alginate with high viscosity (144 cP, condition 1% w / v in water, at 25 °C) and hydroxypropyl-β-cyclodextrin (0.5%, w / v) in deionized water, and stir on a magnetic stirrer at 1 - 4 °C for half an hour / 45 minutes to obtain a homogeneous dispersion. Then, add 5 U of alginate lyase (0.25 U / mg alginate) to the dispersion and mix for 1 minute. Dropwise add this solution into 2% w / v CaCl₂ containing 0.5% w / v hydroxypropyl-β-cyclodextrin, and stir for 15 minutes to obtain Ca 2+ crosslinked alginate microspheres loaded with alginate lyase containing hydroxypropyl-β-cyclodextrin. Wash these microspheres further 3 times with deionized water, 1 minute each time, and expose them to liquid nitrogen for 30 seconds to 2 minutes. Use a freeze dryer set at -57 °C under ultra-high vacuum to lyophilize the frozen microspheres for 24 hours. From Figure 11 (B) and 11(B’), when compared with non-lyophilized microspheres, the freeze-dried or lyophilized microspheres showed no change in shape, thus indicating that the shape of the microspheres was maintained.

[0860] Example 9: Loaded with Cryoprotectants PVP 40kDa (0.5%, w / v) and Trehalose (0.5%, w / v) Freeze-dried Ca of alginate lyase 2+ Degradation of crosslinked alginate microspheres

[0861] Dissolve 1.5% w / v sodium alginate with high viscosity (viscosity 144 cP, condition 1% w / v in water, at 25 °C), 0.5% w / v PVP 40 kDa, and 0.5% w / v trehalose in deionized water, and stir on a magnetic stirrer at 1 - 4 °C for half an hour / 45 minutes to obtain a homogeneous dispersion. Then, add 5 U of alginate lyase (0.25 U / mg alginate) to the dispersion and mix for 1 minute. Dropwise add this solution into 2% w / v CaCl₂ containing 0.5% w / v PVP 40 kDa and 0.5% w / v trehalose, and stir for 15 minutes to obtain Ca 2+ crosslinked alginate microspheres loaded with alginate lyase. Wash these microspheres further with deionized water 3 times, 1 minute each time, and expose them to liquid nitrogen for 30 seconds to 2 minutes. Freeze-dry the frozen microspheres for 24 hours using a freeze-dryer set at -57 °C under ultra-high vacuum. Figure 12 (a) shows the freeze-dried or lyophilized microspheres. Suspend the lyophilized microspheres in 0.01 M phosphate buffer (pH 6.5) and incubate at 37 °C for 72 hours. Degradation of the suspended microspheres was observed, and turbidity was observed, as Figure 12 (c) shows. Figure 12 (e) shows the absorbance spectrum of the degradation products of the lyophilized self-degradable alginate microspheres.

[0862] Example 10: Loaded with Alginate Lyase Containing Cryoprotectant Hydroxypropyl - β - Cyclodextrin (0.5%, w / v) Freeze-dried Ca 2+ Degradation of crosslinked alginate microspheres.

[0863] Dissolve 1.5% w / v sodium alginate with high viscosity (144 cP, condition 1% w / v in water, at 25 °C) and 0.5% w / v hydroxypropyl-β-cyclodextrin in deionized water, and stir on a magnetic stirrer at 1 - 4 °C for half an hour / 45 minutes to obtain a homogeneous dispersion. Then, add 5 U of alginate lyase (0.25 U / mg alginate) to the dispersion and mix for 1 minute. Dropwise add this solution into 2% w / v CaCl₂ containing 0.5% w / v hydroxypropyl-β-cyclodextrin, and stir for 15 minutes to obtain Ca 2+ crosslinked alginate microspheres loaded with alginate lyase. Wash these microspheres further with deionized water 3 times, 1 minute each time, and expose them to liquid nitrogen for 30 seconds to 2 minutes. Freeze-dry the frozen microspheres for 24 hours using a freeze-dryer set at -57 °C under ultra-high vacuum. Figure 12 (b) shows the freeze-dried or lyophilized microspheres. Suspend the lyophilized microspheres in 0.01 M phosphate buffer (pH 6.5) and incubate at 37 °C for 72 hours. Degradation of the suspended microspheres was observed, and turbidity was observed, as Figure 12 (d) shows. Figure 12(e) Showing the absorbance spectrum of the degradation products of freeze-dried biodegradable alginate microspheres.

[0864] Example 11: Ex Vivo Degradation of Alginate Particles Loaded with Alginate Lyase Complexed with Divalent Metal Ions

[0865] In this test, 5 U of alginate lyase (0.25 U / mg of alginate) was loaded into calcium ion-complexed alginate particles and control particles (without enzyme), and they were placed on a liver (bovine) immersed in saline. To evaluate the degradation of the particles, the liver was kept in an oven with the temperature set at 37 ± 1 °C, and the morphological changes of the particles were observed for 48 hours. From Figure 10, the alginate particles loaded with alginate lyase lost their shape within 48 hours, and a film of white residue could be observed. On the other hand, the control particles maintained their shape for 48 hours. A black film on the control particles could be observed, which might be the formation of a biofilm.

[0866] Example 12: In Vitro Biocompatibility of Alginate Particles

[0867] Two different calcium ion-complexed alginate particles loaded with 1 U (0.05 U / mg of alginate) and 5 U (0.25 U / mg of alginate) of alginate lyase were prepared. To evaluate the biocompatibility of the particles, the morphology and viability of cells were observed by optical microscopy, as Figure 13 shown. The cells were seeded in a 24-well plate at a cell density of 10 4 cells / ml. The cells were cultured in α-MEM containing 10% fetal bovine serum and 1% penicillin and streptomycin at 37 °C, 5% CO2, and 95% relative humidity. At least 10 particles with a size of 2 - 3 mm were added to the 24-well plate and incubated for 24 hours. In the control samples, intact particles were observed, and there was no adverse effect on the viability and morphology of osteoblasts. The alginate particles loaded with 5 U of alginate lyase were completely degraded (indicated by the fragments of the degraded alginate particles), while the alginate particles loaded with 1 U of alginate lyase were irregular in shape. The cells were alive and had a flattened morphology under the degraded particles. This data demonstrated the in vitro biocompatibility of calcium-complexed alginate particles loaded with alginate lyase.

[0868] Example 13: From Alginate - Alginate Lyase Precursor Pretreated with Alkaline pH (Carbonate Buffer, pH10) Freeze-dried Ca in solution 2+ Degradation of crosslinked alginate lyase-alginate microspheres

[0869] Dissolve 4% w / v Pronova alginate (G / M < 1, molecular weight approximately 75 kDa - 200 kDa) in carbonate buffer (pH 10 and 0.1 M), and stir on a magnetic stirrer at 1 - 4 °C for half an hour / 45 minutes to obtain a homogeneous dispersion. Then, add 100 mM calcium disodium ethylenediaminetetraacetate hydrate and alginate lyase to obtain a precursor solution containing 2% w / v alginate, 0.05 U / ml (0.0025 U / mg alginate) alginate lyase, and 50 mM calcium disodium ethylenediaminetetraacetate hydrate. Through a microfluidic platform, droplets of the precursor solution with a size of approximately 200 μm are generated using the water-in-oil emulsion technique and exposed to an acidic solution of 2% w / v acetic acid, oil + 0.05% surfactant to crosslink the beads, where Ca 2+ ions are released by the ionization of Ca-EDTA and bind to the eggbox alginate droplets to produce Ca 2+ -crosslinked alginate beads loaded with alginate lyase. To separate the crosslinked alginate beads with a size of approximately 200 μm, the droplets are treated with a droplet disruption solution and further washed with deionized water to remove the oil and surfactant. These beads are further crosslinked in a 2% w / v CaCl2 solution for 5 minutes, and then washed again with deionized water to remove the residual calcium chloride solution. In addition, before being subjected to lyophilization, the beads are stored in an acetate buffer (0.1 M, pH 4.0) containing trehalose and β-cyclodextrin for at least 6 hours. After lyophilization, the beads are rehydrated or reconstituted in a saline solution and evaluated for degradation under static solution conditions for 18 hours. From Figure 14 it can be seen that at T1 hour, some slight decomposition, shape changes with loss of uniformity, and small particle aggregation (< 10 μm) appear in the solution. The trend is followed at T2 hour, with increased surface and shape damage and increased aggregation of small particles in the solution. T18 hours shows a solution completely free of beads, and no aggregation is observed.

[0870] Example 14: Degradation of Reabsorbable Alginate Beads Reconstituted in Saline Solution after Freeze - Drying in an Elastrat Liver Model

[0871] In this article, the lyophilized resorbable alginate beads containing 0.05 U (0.0025 U / mg alginate) alginate lyase are reconstituted in a saline solution and injected into an Elastat liver model to observe the occlusion efficiency of these beads and the degradation of the beads by quantifying the restoration of saline flow. Figure 15 It is described that after injecting the beads into the liver model channel, the flow rate at 0 minutes (T = 0) decreases by > 90% and lasts for 38 minutes. By 50 minutes, 64% of the flow rate is restored, and approximately 2 hours later, the flow rate is restored by 80%. These results indicate that a greater degradation rate is observed under dynamic flow conditions compared to the static conditions given in Example 13.

[0872] Example 15: Degradation behavior of ion-crosslinked resorbable alginate beads in the Elastrat liver model with Ca 2+ ions Evaluation

[0873] The effect of calcium ion crosslinking on the degradation rate of resorbable alginate is shown in Figure 16 . Resorbable alginate beads containing 0.01 U alginate lyase (0.0005 U / mg alginate), which was crosslinked by Ca 2+ ions released by ionizing Ca-EDTA under acidic conditions (0.01, without (w / o) CaCl2), showed poor reduction in saline flow rate after injection into the liver model, indicating deformed alginate beads due to the degradation activity of alginate lyase. By 40 minutes, ~80% of the flow rate was achieved, indicating rapid degradation of the resorbable alginate beads. To reduce the enzyme activity, these beads were further crosslinked in 2% w / v CaCl2 solution for 5 minutes. These particles were injected into the liver model, and a complete reduction in flow rate was observed until 70 minutes, after which an accelerated recovery of flow rate was achieved by 100 minutes. On the other hand, permanent beads (crosslinked only with Ca-ETDA) showed a continuous reduction in flow rate, indicating no degradation or disintegration of the alginate beads was observed over a 140-minute period. These results demonstrate that Ca 2+ crosslinking is dependent on alginate lyase activity and thus controls the degradation of alginate beads.

[0874] Example 16: Alkaline pH - Dependent Reversible Activity of Alginate Lyase

[0875] Alginate lyase and LVG alginate (G / M > 1, molecular weight approximately 75 kDa - 200 kDa) or LVM alginate (G / M < 1, molecular weight approximately 75 kDa - 200 kDa) were dissolved in 0.1 M carbonate buffer (pH 10.0) and 0.01 M phosphate buffer (pH 6.5) at final concentrations of 0.1 U / ml (0.005 U / mg alginate) and 0.1% w / v, respectively. The sample names for the corresponding reactions were LVM- or-LVG-AL pH 10 and LVG-alginate-AL pH 7. Similarly, alginate lyase was pre-incubated in 0.1 M carbonate buffer at pH 10.0 for 15 minutes and then mixed with LVG alginate (G / M > 1, molecular weight approximately 75 kDa - 200 kDa) or LVM alginate (G / M < 1, molecular weight approximately 75 kDa - 200 kDa) in 0.01 M phosphate buffer to achieve an optimal pH 6.5 at final concentrations of 0.1 U / ml and 0.1% w / v, respectively. The sample names for the corresponding reactions were LVM- or-LVG-AL pH 10-7. All the above samples were incubated, and the enzyme activity of alginate lyase was determined by the absorbance of the degradation product at a wavelength of 235 nm ( Figure 17 ).

[0876] Example 17: Effect of Sterilization on Alginate Beads Loaded with 0.05U Alginate Lyase

[0877] The freeze-dried alginate beads containing 0.05 U (0.0025 U / mg alginate) of alginate were sterilized using a 10 kGy electron beam and subjected to self-degradation in a saline solution under physiological conditions (pH 7 and temperature 37 °C) for 24 hours. Figure 18 The presence of alginate lyase activity was demonstrated, which was indicated by an increase in absorbance recorded at 235 nm when compared to a freeze-...

Claims

1. A microsphere capable of self - degrading upon rehydration, comprising: an enzyme pretreated by varying temperature, by varying pH, and / or with a metal ion enzyme inhibitor; and a cross - linked biomaterial; wherein: the cross - linked biomaterial forms a bio - derived microsphere encapsulating the enzyme; and the microsphere is substantially free of water and / or is sterilized.

2. The microsphere according to claim 1, wherein at least one of (i) - (iv) applies: (i) the enzyme is an enzyme that acts on the biomaterial and degrades the biomaterial; (ii) the biomaterial comprises a polysaccharide, a protein, or a glycoprotein; (iii) the microsphere further comprises a photoinitiator, and the biomaterial comprises a photocross - linkable moiety that is photocross - linked; or (iv) the biomaterial is cross - linked by a divalent metal ion.

3. The microsphere according to claim 1 or 2, wherein the self - degradation of the microsphere is controlled by one or more of the following: the pretreatment of the enzyme, the concentration of the enzyme in the microsphere, the enzyme activity, the predetermined molecular weight of the biomaterial, the divalent metal ion used to cross - link the biomaterial, and the amount of the divalent metal ion used to cross - link the biomaterial.

4. The microsphere according to any one of claims 1 - 3, wherein: the enzyme activity is between about 0.0075 U / mg and 0.25 U / mg of biomaterial, and the microsphere degrades within a time period greater than about 20 minutes and less than about 4 hours; the enzyme activity is between about 0.005 U / mg and about ≥0.0025 U / mg of biomaterial, and the microsphere degrades within a time period greater than about 5 days and less than about 30 days; or the enzyme activity is less than about 0.0025 U / mg of biomaterial, and the microsphere degrades within a time period greater than about 30 days.

5. The microsphere according to any one of claims 1 - 4, wherein at least one of (i) - (iv) applies: (i) the residual water content of the microsphere is between about 1% by mass and about 10% by mass; (ii) the microsphere is freeze - dried or dehydrated using supercritical CO2; (iii) the microsphere is sterilized with γ - radiation of about 6 - 10 kGy; or (iv) the microsphere further comprises an anti - inflammatory agent, a chemotherapeutic agent, an antioxidant, a corticosteroid, or a combination thereof.

6. The microspheres according to any one of claims 1-5, wherein: The biomaterial contains alginate and the enzyme is alginate lyase, the biomaterial contains pectin and the enzyme is pectinase, the biomaterial contains hyaluronic acid and the enzyme is hyaluronidase, the biomaterial contains gelatin and the enzyme is matrix metalloproteinase or protease, the biomaterial contains albumin and the enzyme is peptidase, the biomaterial contains collagen and the enzyme is protease, the biomaterial contains fibrinogen and the enzyme is plasmin, the biomaterial contains fibroin and the enzyme is protease, the biomaterial contains starch and the enzyme is amylase, the biomaterial contains chitosan and the enzyme is chitosanase or lysozyme, the biomaterial contains agar / agarose and the enzyme is agarase, the biomaterial contains carrageenan and the enzyme is carrageenase, the biomaterial contains pullulan and the enzyme is pullulanase, the biomaterial contains dextran and the enzyme is dextranase, the biomaterial contains β-glycan and the enzyme is β-glycanase, the biomaterial contains cellulose and the enzyme is cellulase, or the biomaterial contains lignin and the enzyme is ligninase.

7. A method for preparing microspheres capable of self-degrading upon rehydration, the method comprising: forming droplets from a precursor solution, the precursor solution comprising: (i) an enzyme pretreated by varying temperature, by varying pH, and / or with a metal ion enzyme inhibitor; and (ii) a biomaterial; contacting the droplets with a gelling bath comprising a cryoprotectant and a divalent metal ion, thereby crosslinking the biomaterial to form bioderived microspheres encapsulating the enzyme; and dehydrating and optionally sterilizing the microspheres, thereby substantially removing water from the microspheres.

8. The method according to claim 7, wherein at least one of (i)-(vi) applies: (i) the precursor solution further comprises one or more cryoprotectants; (ii) the enzyme is an enzyme that acts on the biomaterial and degrades the biomaterial; (iii) the biomaterial comprises a polysaccharide, a protein, or a glycoprotein; (iv) the microspheres comprise: alginate particles encapsulating alginate lyase, pectin particles encapsulating pectinase, gelatin particles encapsulating matrix metalloproteinase or protease, or carrageenan particles encapsulating carrageenase; (v) the microspheres self-degrade in less than about 4 hours, in a time period greater than about 2 days to less than about 5 days, in a time period greater than about 5 days to less than about 30 days, or in greater than about 30 days; or (vi) the self-degradation of the microspheres is controlled by one or more of: the pretreatment of the enzyme, the concentration of the enzyme in the microspheres, the enzyme activity, the predetermined molecular weight of the biomaterial, the divalent metal ion used to crosslink the biomaterial, and the amount of the divalent metal ion used to crosslink the biomaterial.

9. The method according to claim 7 or 8, wherein: the droplets are in contact with the gelling bath in the range of 10 minutes to 1 hour, and the resulting microspheres degrade in a time period greater than about 20 minutes to less than about 4 hours; The droplet is in contact with the gelling bath for a period ranging from 1 hour to 12 hours, and the resulting microspheres degrade over a time period greater than about 5 days to less than about 30 days; or The droplet is in contact with the gelling bath for a period ranging from 12 hours to 24 hours, and the resulting microspheres degrade over a time period greater than about 30 days.

10. The method according to any one of claims 7 - 9, wherein at least one of (i) - (iv) applies: (i) The residual water content of the microspheres is between about 1% by mass and about 10% by mass; (ii) The dehydration includes freeze - drying the microspheres or drying the microspheres using supercritical CO2; (iii) The sterilization includes irradiating the microspheres with γ - radiation at 6 - 10 kGy; or (iv) The precursor solution and / or the gelling bath further comprises an anti - inflammatory agent, a chemotherapeutic agent, an antioxidant, or a combination thereof.

11. A method for preparing a photopolymerizable microsphere capable of self - degrading upon rehydration, the method comprising: Forming droplets from a precursor solution, the precursor solution comprising: (i) An enzyme pretreated by varying temperature, by varying pH, and / or with a metal ion enzyme inhibitor; (ii) A biomaterial comprising a photocrosslinkable moiety; (iii) A photoinitiator; Irradiating the droplets so that the biomaterial crosslinks to form a photopolymerized bio - derived microsphere encapsulating the enzyme; and Dehydrating the microspheres and optionally sterilizing them to substantially remove water from the microspheres.

12. The method according to claim 11, wherein at least one of (i) - (vi) applies: (i) The photocrosslinkable moiety is selected from acrylate groups, methacrylate groups, vinyl groups, and allyl groups; (ii) The precursor solution further comprises one or more cryoprotectants; (iii) The enzyme is an enzyme that acts on the biomaterial and degrades the biomaterial; (iv) The biomaterial comprises a polysaccharide, a protein, or a glycoprotein; (v) The microspheres self - degrade in less than about 4 hours, over a time period greater than about 2 days to less than about 5 days, over a time period greater than about 5 days to less than about 30 days, or over a time period greater than about 30 days; or (vi) The self - degradation of the microspheres is controlled by one or more of: the pretreatment of the enzyme, the concentration of the enzyme in the microspheres, the enzyme activity, the predetermined molecular weight of the biomaterial, and the amount of time the droplets are irradiated.

13. The method according to claim 11 or 12, wherein the microspheres comprise alginate particles encapsulating alginate lyase, pectin particles encapsulating pectinase, hyaluronic acid particles encapsulating hyaluronidase, gelatin particles encapsulating matrix metalloproteinase or protease, albumin particles encapsulating peptidase, collagen particles encapsulating protease, fibrinogen particles encapsulating plasmin, silk fibroin particles encapsulating protease, starch particles encapsulating amylase, chitosan particles encapsulating chitosanase or lysozyme, agar / agarose particles encapsulating agarase, carrageenan particles encapsulating carrageenase, pullulan particles encapsulating pullulanase, dextran particles encapsulating dextranase, β - glycan particles encapsulating β - glycanase, cellulose particles encapsulating cellulase, or lignin particles encapsulating ligninase.

14. The method according to any one of claims 11 - 13, wherein at least one of (i) - (iv) applies: (i) The residual water content of the microspheres is between about 1% by mass and about 10% by mass; (ii) The dehydration includes freeze - drying the microspheres or drying the microspheres using supercritical CO2; (iii) The sterilization includes irradiating the microspheres with γ - radiation at 6 - 10 kGy; or (iv) The precursor solution further comprises an anti - inflammatory agent, a chemotherapeutic agent, an antioxidant, or a combination thereof.

15. A method for preparing microspheres capable of self - degrading upon rehydration, the method comprising: Forming droplets from a precursor solution, the precursor solution comprising: (i) A biomaterial comprising covalently cross - linkable moieties; and (ii) A homobifunctional cross - linker or a heterobifunctional cross - linker; Covalently cross - linking the biomaterial to form bio - derived microspheres; Swelling an enzyme pretreated by varying temperature, by varying pH, and / or with a metal ion enzyme inhibitor into the microspheres such that the bio - derived microspheres encapsulate the enzyme; and Dehydrating the microspheres and optionally sterilizing them, thereby substantially removing water from the microspheres.

16. The method according to claim 15, wherein at least one of (i) - (vi) applies: (i) The covalently cross - linkable moieties comprise amino groups or carboxyl groups; (ii) The precursor solution further comprises one or more cryoprotectants; (iii) The enzyme is an enzyme that acts on the biomaterial and degrades the biomaterial; (iv) The biomaterial comprises a polysaccharide, a protein, or a glycoprotein; (v) The microspheres self - degrade in less than about 4 hours, in a time period greater than about 2 days to less than about 5 days, in a time period greater than about 5 days to less than about 30 days, or in greater than about 30 days; or (vi) The self - degradation of the microspheres is controlled by one or more of: the pretreatment of the enzyme, the concentration of the enzyme in the microspheres, the enzyme activity, the predetermined molecular weight of the biomaterial, and the homobifunctional cross - linker or heterobifunctional cross - linker used to cross - link the biomaterial.

17. The method according to claim 15 or 16, wherein the microspheres comprise alginate particles encapsulating alginate lyase, pectin particles encapsulating pectinase, hyaluronic acid particles encapsulating hyaluronidase, gelatin particles encapsulating matrix metalloproteinase or protease, albumin particles encapsulating peptidase, collagen particles encapsulating protease, fibrinogen particles encapsulating plasmin, silk fibroin particles encapsulating protease, starch particles encapsulating amylase, chitosan particles encapsulating chitosanase or lysozyme, agar / agarose particles encapsulating agarase, carrageenan particles encapsulating carrageenase, pullulan particles encapsulating pullulanase, dextran particles encapsulating dextranase, β - glycan particles encapsulating β - glycanase, cellulose particles encapsulating cellulase, or lignin particles encapsulating ligninase.

18. The method according to any one of claims 15 - 17, wherein at least one of (i) - (iv) applies: (i) The residual water content of the microspheres is between about 1% by mass and about 10% by mass; (ii) The dehydration includes freeze - drying the microspheres or drying the microspheres using supercritical CO2; (iii) The sterilization includes irradiating the microspheres with γ-radiation of 6 - 10 kGy; or (iv) The precursor solution further comprises an anti-inflammatory agent, a chemotherapeutic agent, an antioxidant, or a combination thereof.

19. A method for preparing microspheres capable of self-degrading upon rehydration, the method comprising: Forming droplets from a precursor solution, the precursor solution comprising: (i) A biomaterial comprising covalently crosslinkable moieties; (ii) An enzyme that has been pretreated by varying temperature, by varying pH, and / or with a metal ion enzyme inhibitor; and (iii) A homobifunctional crosslinker or a heterobifunctional crosslinker; Covalently crosslinking the biomaterial to form a bio-derived microsphere encapsulating the enzyme; and Dehydrating the microspheres and optionally sterilizing them, thereby substantially removing water from the microspheres.

20. The method according to claim 19, wherein at least one of (i)-(vi) applies: (i) The covalently crosslinkable moieties comprise amino groups or carboxyl groups; (ii) The precursor solution further comprises one or more cryoprotectants; (iii) The enzyme is an enzyme that acts on the biomaterial and degrades the biomaterial; (iv) The biomaterial comprises a polysaccharide, a protein, or a glycoprotein; or (v) The microspheres self-degrade in less than about 4 hours, in a time period greater than about 2 days to less than about 5 days, in a time period greater than about 5 days to less than about 30 days, or in greater than about 30 days; or (vi) The self-degradation of the microspheres is controlled by one or more of: the pretreatment of the enzyme, the concentration of the enzyme in the microspheres, the enzyme activity, the predetermined molecular weight of the biomaterial, and the homobifunctional crosslinker or heterobifunctional crosslinker used to crosslink the biomaterial.

21. The method according to claim 19 or 20, wherein the microspheres comprise alginate particles encapsulating alginate lyase, pectin particles encapsulating pectinase, hyaluronic acid particles encapsulating hyaluronidase, gelatin particles encapsulating matrix metalloproteinase or protease, albumin particles encapsulating peptidase, collagen particles encapsulating protease, fibrinogen particles encapsulating plasmin, silk fibroin particles encapsulating protease, starch particles encapsulating amylase, chitosan particles encapsulating chitosanase or lysozyme, agar / agarose particles encapsulating agarase, carrageenan particles encapsulating carrageenase, pullulan particles encapsulating pullulanase, dextran particles encapsulating dextranase, β-glycan particles encapsulating β-glycanase, cellulose particles encapsulating cellulase, or lignin particles encapsulating ligninase.

22. The method according to any one of claims 19 - 21, wherein at least one of (i)-(iv) applies: (i) The residual water content of the microspheres is between about 1% mass and about 10% mass; (ii) The dehydration comprises freeze-drying the microspheres or drying the microspheres using supercritical CO2; (iii) The sterilization includes irradiating the microspheres with γ-radiation of 6 - 10 kGy; or (iv) The precursor solution further comprises an anti-inflammatory agent, a chemotherapeutic agent, an antioxidant, or a combination thereof.

23. A method for preparing thermogelating microspheres capable of self-degrading upon rehydration, the method comprising: Heat a precursor solution containing a biocompatible material to melt the biocompatible material; Add an enzyme pretreated by varying temperature, varying pH, and / or with a metal ion enzyme inhibitor to the precursor solution; Form droplets from the precursor solution; Cool the droplets to form thermogelated bioderived microspheres encapsulating the enzyme; and Dehydrate the microspheres and optionally sterilize them to substantially remove water from the microspheres.

24. The method according to claim 23, wherein at least one of (i)-(v) applies: (i) The precursor solution further comprises one or more cryoprotectants; (ii) The enzyme is an enzyme that acts on and degrades the biocompatible material; (iii) The biocompatible material comprises a polysaccharide, protein, or glycoprotein; (iv) The microspheres autodegrade in less than about 4 hours, in a time period greater than about 2 days to less than about 5 days, in a time period greater than about 5 days to less than about 30 days, or in greater than about 30 days; or (v) The autodegradation of the microspheres is controlled by one or more of: pretreatment of the enzyme, concentration of the enzyme in the microspheres, enzyme activity, and a predetermined molecular weight of the biocompatible material.

25. The method according to claim 23 or 24, wherein the microspheres comprise pectin particles encapsulating pectinase, gelatin particles encapsulating matrix metalloproteinase or protease, albumin particles encapsulating peptidase, collagen particles encapsulating protease, fibrinogen particles encapsulating plasmin, silk fibroin particles encapsulating protease, starch particles encapsulating amylase, chitosan particles encapsulating chitosanase or lysozyme, or agar / agarose particles encapsulating agarase.

26. The method according to any one of claims 23-25, wherein at least one of (i)-(iv) applies: (i) The residual water content of the microspheres is between about 1% by mass and about 10% by mass; (ii) The dehydration comprises freeze-drying the microspheres or drying the microspheres using supercritical CO2; (iii) The sterilization comprises irradiating the microspheres with γ-radiation at 6-10 kGy; or (iv) The precursor solution further comprises an anti-inflammatory agent, a chemotherapeutic agent, an antioxidant, or a combination thereof.

27. A method of preparing microspheres capable of autodegrading, the method comprising: Forming a precursor solution comprising: (i) an enzyme; and (ii) a biocompatible material; Passing the precursor solution through a needle under the influence of an electrostatic potential to form droplets; and Contacting the droplets with a gelling bath comprising divalent metal ions to crosslink the biocompatible material to form bioderived microspheres encapsulating the enzyme.

28. The method according to claim 27, further comprising dehydrating the microspheres and optionally sterilizing them to substantially remove water from the microspheres to form microspheres capable of autodegrading upon rehydration.

29. The method according to claim 27 or 28, wherein at least one of (i)-(v) applies: (i) The precursor solution further comprises one or more cryoprotectants; (ii) The enzyme is an enzyme that acts on and degrades the biocompatible material; (iii) The biocompatible material comprises a polysaccharide, protein, or glycoprotein; (iv) the microspheres autodegrade in less than about 4 hours, in a time period greater than about 2 days to less than about 5 days, in a time period greater than about 5 days to less than about 30 days, or in greater than about 30 days; or (v) the autodegradation of the microspheres is controlled by one or more of the following: pretreatment of the enzyme, concentration of the enzyme in the microspheres, enzyme activity, predetermined molecular weight of the biomaterial, divalent metal ions used for crosslinking the biomaterial, and amount of divalent metal ions used for crosslinking the biomaterial.

30. The method according to any one of claims 27-29, wherein the microspheres comprise: alginate particles encapsulating alginate lyase, pectin particles encapsulating pectinase, hyaluronic acid particles encapsulating hyaluronidase, gelatin particles encapsulating matrix metalloproteinase or protease, albumin particles encapsulating peptidase, collagen particles encapsulating protease, fibrinogen particles encapsulating plasmin, fibroin particles encapsulating protease, starch particles encapsulating amylase, chitosan particles encapsulating chitosanase or lysozyme, agar / agarose particles encapsulating agarase, carrageenan particles encapsulating carrageenase, pullulan particles encapsulating pullulanase, dextran particles encapsulating dextranase, b-glycan particles encapsulating b-glycanase, cellulose particles encapsulating cellulase, or lignin particles encapsulating ligninase.

31. The method according to any one of claims 28-30, wherein at least one of (i)-(iv) applies: (i) the residual water content of the microspheres is between about 1% mass and about 10% mass; (ii) the dehydration comprises freeze-drying the microspheres or drying the microspheres using supercritical CO2; (iii) the sterilization comprises irradiating the microspheres with γ-radiation of 6-10 kGy; or (iv) the precursor solution and / or the gelling bath further comprises an anti-inflammatory agent, a chemotherapeutic agent, an antioxidant, or a combination thereof.

32. A method for inducing autodegradable embolization in a subject in need thereof, comprising administering a plurality of microspheres according to any one of claims 1-6 into a blood vessel of the subject.

33. The method according to claim 32, wherein the blood vessel is the geniculate artery and / or the method induces prostatic artery embolization, induces uterine artery embolization, or the microspheres comprise a chemotherapeutic agent or are mixed with a chemotherapeutic agent, and the method induces transarterial chemoembolization (TACE).

34. A method for treating a disease or disorder in a subject in need thereof, comprising administering to the subject a plurality of microspheres according to any one of claims 1-6.

35. The method according to claim 34, wherein the disease or disorder is selected from tendinopathy, osteoarthritis, frozen shoulder, tennis elbow (lateral epicondylitis), golfer's elbow (medial epicondylopathy), pitcher's elbow (flexor tendinitis), Achilles tendinopathy, plantar fasciitis, symptomatic accessory navicular pain, hamstring tendinopathy, jumper's knee (patellar tendinitis), runner's knee (patellofemoral pain syndrome (PFPS)), pes anserine bursitis (knee pain), posterior tibial tendonopathy, wrist (TFCC - triangular fibrocartilage complex) tendonopathy, trigger finger (stenosing flexor tenosynovitis), and hemarthrosis.

36. A method for rapidly degrading microspheres in a subject, comprising administering an emergency rescue solution to the subject, wherein a plurality of microspheres according to any one of claims 1-6 have been previously administered to the subject, and the emergency rescue solution comprises an enzyme capable of degrading the microspheres.

37. The method according to claim 36, wherein the enzyme is complementary to the biomaterial used to form the plurality of microspheres.

38. The method according to claim 36 or 37, wherein: the microspheres comprise alginate particles encapsulating alginate lyase and the enzyme is alginate lyase, the microspheres comprise pectin particles encapsulating pectinase and the enzyme is pectinase, the microspheres comprise hyaluronic acid particles encapsulating hyaluronidase and the enzyme is hyaluronidase, the microspheres comprise gelatin particles encapsulating matrix metalloproteinase or protease and the enzyme is matrix metalloproteinase or protease, the microspheres comprise albumin particles encapsulating peptidase and the enzyme is peptidase, the microspheres comprise collagen particles encapsulating protease and the enzyme is protease, the microspheres comprise fibrinogen particles encapsulating plasmin and the enzyme is plasmin, the microspheres comprise silk fibroin particles encapsulating protease and the enzyme is protease, the microspheres comprise starch particles encapsulating amylase and the enzyme is amylase, the microspheres comprise chitosan particles encapsulating chitosanase or lysozyme and the enzyme is chitosanase or lysozyme, the microspheres comprise agar / agarose particles encapsulating agarase and the enzyme is agarase, the microspheres comprise carrageenan particles encapsulating carrageenase and the enzyme is carrageenase, the microspheres comprise pullulan particles encapsulating pullulanase and the enzyme is pullulanase, the microspheres comprise dextran particles encapsulating dextranase and the enzyme is dextranase, the microspheres comprise β-glycan particles encapsulating β-glycanase and the enzyme is β-glycanase, the microspheres comprise cellulose particles encapsulating cellulase and the enzyme is cellulase, or the microspheres comprise lignin particles encapsulating ligninase and the enzyme is ligninase.

39. The method according to any one of claims 36-38, wherein the emergency rescue solution further comprises a divalent metal chelator.

40. A method for rapidly degrading divalent metal ion-crosslinked microspheres in a subject, comprising administering an emergency rescue solution to the subject, wherein a plurality of divalent metal ion-crosslinked microspheres according to any one of claims 1-6 have been previously administered to the subject, and the emergency rescue solution comprises an anion, a phosphate buffer, or a combination thereof.

41. The method according to claim 40, wherein at least one of (i)-(iii) applies: (i) the anion comprises citrate; (ii) the phosphate buffer comprises phosphate buffered saline; and (iii) the microspheres comprise alginate particles encapsulating alginate lyase, pectin particles encapsulating pectinase, or carrageenan particles encapsulating carrageenase.

42. The method according to claim 40 or 41, wherein the microspheres comprise alginate particles encapsulating alginate lyase, pectin particles encapsulating pectinase, or carrageenan particles encapsulating carrageenase; and wherein the anion comprises citrate; or wherein the microspheres comprise alginate particles encapsulating alginate lyase, pectin particles encapsulating pectinase, or carrageenan particles encapsulating carrageenase; and wherein the phosphate buffer comprises phosphate buffered saline.

43. A kit, comprising: (i) a plurality of microspheres according to any one of claims 1 - 6; and an enzyme capable of rapidly degrading the microspheres when dissolved to form a solution; or (ii) a plurality of divalent metal ion crosslinked microspheres according to any one of claims 1 - 6; and an inorganic salt capable of rapidly degrading the microspheres when dissolved to form a solution.

44. The kit according to claim 43, wherein the enzyme in (i) is complementary to the biomaterial used to form the plurality of microspheres.

45. The kit according to claim 43 or 44, wherein for the plurality of microspheres in (i): the microspheres comprise alginate particles encapsulating alginate lyase and the enzyme is alginate lyase, the microspheres comprise pectin particles encapsulating pectinase and the enzyme is pectinase, the microspheres comprise hyaluronic acid particles encapsulating hyaluronidase and the enzyme is hyaluronidase, the microspheres comprise gelatin particles encapsulating matrix metalloproteinase or protease and the enzyme is matrix metalloproteinase or protease, the microspheres comprise albumin particles encapsulating peptidase and the enzyme is peptidase, the microspheres comprise collagen particles encapsulating protease and the enzyme is protease, the microspheres comprise fibrinogen particles encapsulating plasmin and the enzyme is plasmin, the microspheres comprise fibroin particles encapsulating protease and the enzyme is protease, the microspheres comprise starch particles encapsulating amylase and the enzyme is amylase, the microspheres comprise chitosan particles encapsulating chitosanase or lysozyme and the enzyme is chitosanase or lysozyme, the microspheres comprise agar / agarose particles encapsulating agarase and the enzyme is agarase, the microspheres comprise carrageenan particles encapsulating carrageenase and the enzyme is carrageenase, the microspheres comprise pullulan particles encapsulating pullulanase and the enzyme is pullulanase, the microspheres comprise dextran particles encapsulating dextranase and the enzyme is dextranase, the microspheres comprise β - glycan particles encapsulating β - glycanase and the enzyme is β - glycanase, the microspheres comprise cellulose particles encapsulating cellulase and the enzyme is cellulase, or the microspheres comprise lignin particles encapsulating ligninase and the enzyme is ligninase.

46. The kit according to claim 43, wherein For the plurality of microspheres in (ii): the microspheres comprise alginate particles encapsulating alginate lyase, pectin particles encapsulating pectinase, or carrageenan particles encapsulating carrageenase.

47. The kit according to claim 43 or 46, wherein the inorganic salt in (ii) releases citrate or phosphate when dissolved to form a solution.

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