WO2017101883A1 - 一种可在生理条件下降解的水凝胶 - Google Patents

一种可在生理条件下降解的水凝胶 Download PDF

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WO2017101883A1
WO2017101883A1 PCT/CN2016/110876 CN2016110876W WO2017101883A1 WO 2017101883 A1 WO2017101883 A1 WO 2017101883A1 CN 2016110876 W CN2016110876 W CN 2016110876W WO 2017101883 A1 WO2017101883 A1 WO 2017101883A1
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group
alkyl
hydrogel
functional group
linking
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French (fr)
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韩捷
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韩捷
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Priority to US16/063,687 priority Critical patent/US11285103B2/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6903—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel

Definitions

  • the present invention relates to the field of bioengineering and, in particular, to a hydrogel that can degrade under physiological conditions.
  • Hydrogels are potential drug delivery system materials.
  • the high water content of the hydrogel renders the hydrogel biocompatible, reducing the inflammatory response of the tissue with which it is in contact.
  • Hydrogels are suitable for small molecule and macromolecular drugs.
  • Hydrogels provide a sufficiently aqueous environment, especially suitable for maintaining the biological activity and structural integrity of peptides, proteins, low (poly)nucleotides or poly(poly)nucleotide drugs. .
  • the hydrogel loaded with the bioactive substance can form a depot in the patient after administration to release the biologically active substance for a desired length of time.
  • a hydrogel-based reservoir there are two methods for preparing a hydrogel-based reservoir: a non-covalent and covalent reservoir.
  • a biologically active substance such as a drug is encapsulated in a hydrogel by a physical method rather than by a chemical bond.
  • the average pore size in the three-dimensional network of hydrogels must be less than the size of the bioactive material to ensure efficient encapsulation of the hydrogel. Therefore, the biologically active substance cannot be added after the hydrogel is formed.
  • the hydrogel In non-covalent methods, the hydrogel must be chemically crosslinked in the presence of a biologically active material or, in the presence of a biologically active material, formed by physical cross-linking of the self-assembly process. Hydrogels can be prepared by crosslinking hydrophilic biopolymers or synthetic polymers.
  • hydrogel formed by chemical or physical crosslinking of the synthetic polymer include polyethylene glycol, polypropylene glycol, poly(lactic-ethylene glycol copolymer) acid (PLGA) polymer, and the like.
  • PLGA poly(lactic-ethylene glycol copolymer) acid
  • the biologically active substance is attached to the hydrogel via a reversible or degradable linking group.
  • the ester bond commonly used as a biodegradable bond may spontaneously hydrolyze in a pure buffer at physiological pH 7.4 without enzyme.
  • Many hydrogels contain a large number of ester bonds in order to effectively release biologically active substances. Both local high density ester linkages and tightly packed bioactive materials can cause side reactions.
  • the amino group in the biologically active substance is likely to be in a position close to the ester bond, and the amino group provides a nucleophilic agent to promote ester bond cleavage and subsequent amidation. This process results in a very stable amide bond between the bioactive material and the polymer.
  • the biologically active substance is not released until the polymer chain to which it is attached is degraded, and the biologically active substance is permanently modified. Such modifications are known to reduce the biological activity of biologically active substances and may cause side effects. In addition, this undesirable modification process is largely uncontrollable, resulting in a variety of molecular structures.
  • hydrogel After the administration is completed, the hydrogel needs to be removed from the body. Removal of hydrogels by surgery often adds pain to the patient. Although some known hydrogels claim to be biodegradable, the degradation is uncontrolled and therefore unpredictable.
  • the present invention introduces a degradable linking group within the hydrogel backbone or within the crosslink between the hydrogel backbones.
  • These degradable linking groups are capable of being automatically cleaved by chemical reactions under physiological conditions (eg, 37 ° C, pH 7.0-7.6), depending on temperature and pH, without the aid of enzymes or other reagents. Since physiological conditions are generally constant in the human population, this novel hydrogel can biodegrade at a predetermined rate, with little difference between administration and individual patient, resulting in a more consistent therapeutic effect. In addition, the new hydrogel can be completely degraded into small molecular weight fragments, which are removed from the body and do not accumulate at the injection site or in the body.
  • the present invention provides a hydrogel which is degraded to a water-soluble, smaller molecular weight component by a non-enzymatic process under physiological conditions, and a method of preparing the hydrogel.
  • the backbone or crosslinker of the hydrogel contains degradable linking groups that cleave under biological conditions, resulting in degradation of the hydrogel.
  • the invention also relates to degradable linking groups per se, as well as intermediates in the formation of the invention.
  • the degradable linking group of the present invention includes a moiety which can be automatically cleaved under physiological conditions, and a reactive group which forms a covalent bond with the reactive polymer.
  • the reactive polymer in the present invention may be a hydrogel skeleton or a crosslinked product.
  • the portion that can be automatically broken under physiological conditions has a formula
  • X, Y, Y 1 , A, B, and J 1 are defined in detail below.
  • Y, A, and B contain at least one reactive group capable of forming a covalent bond with the reactive polymer.
  • the wavy line is the point of attachment of the degradable linking group to the bioactive material or reactive polymer to form an amide bond or an ester bond.
  • a degradable linking group links two or more reactive polymers together.
  • the hydrogel prepared according to the present invention can be applied to various fields such as drug delivery systems, biomedical engineering, and the like.
  • Hydrogels prepared in accordance with the present invention may also comprise one or more drugs or biologically active substances.
  • the one or more drugs or biologically active substances may be encapsulated within the hydrogel by physical means rather than by chemical bonding.
  • the drug or biologically active substance may also be attached to one end of the degradable linking group, and the other end of the degradable linking group is coupled to the hydrogel. After the degradable linking group is broken, the drug or biologically active substance can be released independently without depending on the degradation of the hydrogel.
  • the present invention also provides a process for the preparation of a biodegradable hydrogel, wherein the rate of drug release and the rate of degradation of the hydrogel are controllable.
  • Figure 1 shows the changes in blood glucose in rats within one week after injection of physiological saline, insulin detemir and insulin hydrogel into diabetic rats.
  • the hydrogel of the present invention is a polymer obtained by crosslinking a degradable linking group under physiological conditions. At physiological pH and temperature, the hydrogel loses structural integrity due to cleavage of these linking groups, and the resulting fragments are small enough and soluble to be cleared from the body by conventional biological pathways.
  • the "biologically active substance" in the present invention means a therapeutic or pharmacological substance.
  • the biologically active substance may be a small molecule, a polypeptide, a protein, a DNA, an RNA, a cell or the like.
  • biologically active substances include, but are not limited to, the following therapeutic fields: ACE inhibitors; anti-angina drugs; antiarrhythmic drugs; anti-asthmatic drugs; anticholesterol drugs; anticonvulsants; antidepressants; antidiarrheal preparations; Histamine; antihypertensive; anti-infective; anti-inflammatory; anti-lipid; anti-manic drug; anti-nausea; anti-thyroid; anti-thyroid; anti-tumor; antitussive; anti- uric acid; Antiviral agent; acne medicine; alkaloid; amino acid preparation; anabolic drug; analgesic; anesthetic; angiogenesis inhibitor; antacid; anti-arthritis drug; antibiotic; anticoagulant;
  • biodegradable or “automatically cleavable” means degradation under physiological conditions without enzymatic catalysis.
  • bonds include, but are not limited to, acetal bonds, ester bonds, imine bonds, hydrazine bonds, carboxylic anhydrides, aconite bonds, orthoester bonds, maleamic acid amide bonds, phosphoramide bonds, phosphoryl ester bonds, a sulfonate bond, an aromatic urethane bond, a combination thereof, and the like.
  • Preferred are carboxylate linkages and carbonate linkages.
  • the common disadvantage of these bonds is their poor stability, which is easily degraded by various enzymes and is also prone to hydrolysis. The drug release rate and hydrogel degradation rate of medicinal hydrogels based on these bonds cannot be accurately controlled.
  • Amide bonds are known for their stability, and hydrolysis typically requires a strong acid (such as sulfuric acid, hydrochloric acid) or a strong base (such as sodium hydroxide) and a high temperature (e.g., 100 ° C). Due to the relative stability of the amide in vivo, an amide structure that can be automatically cleaved without enzymatic hydrolysis is very rare.
  • the present invention shows that by virtue of the advantages of intramolecular chemical catalysis, the amide bond can be hydrolyzed under mild conditions. Designing and modifying the appropriate molecular backbone, as well as selecting the space, electron-withdrawing or electron-donating substituents at different positions of the backbone, can modulate the rate of reaction of the amide bond under physiological conditions. The same principle applies to ester bonds.
  • the portion of the degradable linking group of the present invention which is cleaved under physiological conditions can be represented by the following formula:
  • X is selected from -OH or -HN-R 0 ;
  • Y is selected from:
  • Y 1 is selected from
  • J 1 is -C (R 10 R 11) or a covalent bond.
  • a and B together form a ring system comprising: an aryl group having 6 to 15 carbon atoms; a cycloalkyl group having a 4, 5, 6, 7, 8, 9 or 10 membered ring; having 4, 5, 6, 7 , 8, 9, or 10 membered cycloalkenyl; cycloalkynyl having 5, 6, 7, 8, 9 or 10 membered rings; saturated and unsaturated monocyclic, polycyclic and fused rings; saturated and unsaturated Monoheterocyclic, polyheterocyclic and fused heterocyclic rings each containing one or more heterocyclic atoms N, S or O, and each ring is a 4- to 5-membered ring.
  • Each of the foregoing rings is optionally substituted with one or more groups selected from the group consisting of lower alkyl, lower alkoxy, acyl, acyloxy, alkoxycarbonyl, aryl , phenyl, benzyl, halogen, haloformyl, halo lower alkyl, trifluoromethyl, trifluoromethoxy, trichloromethyl, cyano, isocyanide, isocyanate (or salt) , isothiocyanate, thiocyanate (or salt), lower alkylthio, amino, imino, amino lower alkyl, lower alkylamino, lower alkane Lower dialkylamino, hydroxy, hydroxyalkyl, nitro, nitrile, isonitrile, pyridyl, azide, carboxyl, carboxamido, acetate, thiolalkyl, carbonate (or salt ), carbamate, lower alkylcarbamyl, dilower alky
  • A, B and the atoms to which they are attached form an aromatic ring or an aromatic heterocyclic ring, optionally substituted by at least one group defined above; or A, B and the atoms to which they are attached form a polyaromatic ring or A polyaromatic heterocycle, optionally substituted with at least one group as defined above.
  • Examples of various rings include, but are not limited to, acridine, azepane, azepine, azocane, benzofuran, benzimidazole, benzothiophene, benzo[c]thiophene.
  • Y 1 (or Y, if Y 1 is a covalent bond) and J 1 (or C(O), if J 1 is a covalent bond) are respectively linked to two atoms that form the same ring and are separated by a bond. .
  • R 0 , R O , R 3 , R 4 , R 10 , R 11 , R n , R p , R q are each independently selected from a hydrogen atom, a lower alkyl group, a lower alkoxy group, an acyl group, an acyloxy group, an alkoxy group.
  • R 0 , R O , R 3 , R 4 , R 10 , R 11 , R n , R p , R q are each independently selected from -SO 2 -OH, -SO 2 -NR m1 R m2 , -SO 2 - R m3 , -OR m4 , -SR m5 , -NR m6 R m7 , -C(O)R m8 , -C(O)OR m9 , -OC(O)R m10 , -NHC(O)R m11 ,- C(O)NR m12 R m13 , -NHC(O)NR m14 R m15 , where R m1 , R m2 , R m3 , R m4 , R m5 , R m6 , R m7 , R m8 , R m9 , R m10 , R m11
  • R p and R q are each independently selected from C 1 -C 20 alkyl, (C 1 -C 10 alkyl)OH, (C 1 -C 10 alkyl)SH, (C 2 -C 3 alkyl)SCH 3 , (C 1 -C 4 alkyl)CONH 2 , (C 1 -C 10 alkyl)COOH, (C 1 -C 10 alkyl)NH 2 , (C 1 -C 4 alkyl)NHC (NH 2 + ) NH 2 , (C 0 -C 4 alkyl) (C 3 -C 6 cycloalkyl), (C 0 -C 4 alkyl) (C 2 -C 5 heterocyclic), (C 0 -C 4 Alkyl)(C 6 -C 10 aryl), (C 0 -C 4 alkyl)(C 6 -C 10 aryl)R 16 ,(C 1 -C 4 alkyl)(C 3 -C 9 hetero Aryl), C
  • the degradable portion of the degradable linking group in the present invention is degraded by an intramolecular cyclization reaction.
  • a portion of the biologically active substance R 14 and R 15 may be a nitrogen-containing or hydroxy group, the reaction may also be part of a nitrogen-containing polymer or a hydroxy group.
  • Degradation by the intramolecular cyclization reaction does not require the participation of enzymes, the rate being dependent on temperature and physiological pH. Since the body temperature and physiological pH are basically the same in the population, this degradation mode eliminates the differences between humans and individuals that occur in enzymatic degradation, resulting in a more consistent and reproducible therapeutic effect.
  • J 1 is C(R 10 R 11 ), Y 1 is a covalent bond, and the chemical formula of the degradable linking group is as follows:
  • Y 1 is C(R 3 R 4 ), J 1 is a covalent bond, and the chemical formula of the degradable linking group is as follows:
  • Y 1 is C(R 3 R 4 )
  • J 1 is C(R 10 R 11 )
  • the chemical formula of the degradable linking group is as follows:
  • the chemical formula of the degradable linking group is as follows:
  • the chemical formula of a class of degradable linking groups is as follows:
  • X is selected from -OH or -HN-R 0 ;
  • Y is selected from:
  • Y 1 is selected from
  • J 1 is -C(R 10 R 11 ) or a covalent bond.
  • a, b and the atoms attached thereto form a bicyclic system, including a saturated 5-membered ring or a 6-membered ring and an aromatic ring fused thereto.
  • the saturated 5-membered or 6-membered ring may be a cycloalkyl group or a heterocyclic ring containing one or more heterocyclic atoms N, S or O.
  • Y 1 (or Y, if Y 1 is a covalent bond) and J 1 (or C(O), if J 1 is a covalent bond) are separated from each other by a saturated 5-membered ring or a 6-membered ring. The two atoms are connected.
  • the aromatic ring is not directly bonded to J, Y, C(O), or Y.
  • the hydrogen atom of the aromatic ring is optionally substituted by one or more groups selected from the group consisting of lower alkyl, lower alkoxy, acyl, acyloxy, alkoxycarbonyl, aryl, phenyl, benzyl, halogen.
  • n are integers of 0 or 1, respectively, but m and n are not 0 at the same time.
  • a class of degradable linking groups has the formula: wherein X, Y, R 3 , R 4 , R 10 , R 11 , a and b are as defined above:
  • a, b and the atom to which it is attached form a single aromatic ring, a polyaromatic ring, a fused aromatic ring, a monoheteroaryl ring, a polyheteroaryl ring or a fused heteroaryl ring, optionally substituted by one or more groups,
  • the group is selected from the group consisting of lower alkyl, lower alkoxy, acyl, acyloxy, alkoxycarbonyl, aryl, phenyl, benzyl, halogen, haloformyl, halo lower alkyl, trifluoromethyl, tri Fluoromethoxy, trichloromethyl, cyano, isocyanide, isocyanate (or salt), isothiocyanate, thiocyanate (or salt), lower alkylthio, amino, imino, amino lower alkane Base, lower alkylamino, lower alkylamino, hydroxy, hydroxyalkyl, nitro, nit
  • a class of degradable linking groups comprising a 6-membered ring has the following chemical formula:
  • U 1 , U 2 , U 3 , U 4 , U 5 and U 6 are independently selected from CR 12 and N;
  • X is selected from -OH or -HN-R 0 ;
  • Y is selected from:
  • Y 1 is selected from
  • J 1 is -C(R 10 R 11 ) or a covalent bond.
  • R 12 is selected from the group consisting of a hydrogen atom, a lower alkyl group, a lower alkoxy group, an acyl group, an acyloxy group, an alkoxycarbonyl group, an aryl group, a phenyl group, a benzyl group, a halogen group, a halogenoformyl group, a halogenated lower alkyl group, and a trifluoro group.
  • R 12 is selected from -SO 2 -OH, -SO 2 -NR m1 R m2 , -SO 2 -R m3 , -OR m4 , -SR m5 , -NR m6 R m7 , -C(O)R m8 ,- C(O)OR m9 , -OC(O)R m10 , -NHC(O)R m11 , -C(O)NR m12 R m13 , -NHC(O)NR m14 R m15 , where R m1 , R m2 , R m3 , R m4 , R m5 , R m6 , R m7 , R m8 , R m9 , R m10 , R m11 , R m12 , R m13 , R m14 and R m15 are independently selected from a hydrogen atom (H), (C 1
  • R 12 comprises a functional group such as a hydroxyl group, an amino group, a carboxyl group, a thiol group, and the like, and can form a covalent bond with a crosslinking functional group of the reactive polymer.
  • R 12 comprises an azide or alkynyl group and can be reacted by a click chemistry with a corresponding alkynyl or azide on the reactive polymer.
  • a class of degradable linking groups comprising a 6-membered ring has the following chemical formula:
  • X is selected from -OH or -HN-R 0 ;
  • Y is selected from:
  • Introducing groups in this class include, but are not limited to, the following examples:
  • Y in the above three formulas is selected from the group consisting of NH, NR O , O (provided that X is not OH or SH).
  • Y is NR O and X is HN-R 0 or OH.
  • the chemical formula of this type of degradable linking group is as follows:
  • R O is a hydrogen atom (H), and the chemical formula of such a degradable linking group is as follows:
  • R 5 , R 6 , R 7 and R 8 are independently selected from the group consisting of a hydrogen atom, a lower alkyl group, a lower alkoxy group, an acyl group, an acyloxy group, an alkoxycarbonyl group, an aryl group, a phenyl group, a benzyl group, a halogen group, a halogenated group A.
  • R 5 , R 6 , R 7 and R 8 are independently selected from -SO 2 -OH, -SO 2 -NR m1 R m2 , -SO 2 -R m3 , -OR m4 , -SR m5 , -NR m6 R m7 , -C(O)R m8 , -C(O)OR m9 , -OC(O)R m10 , -NHC(O)R m11 , -C(O)NR m12 R m13 ,-NHC(O)NR m14 R m15 , wherein R m1 , R m2 , R m3 , R m4 , R m5 , R m6 , R m7 , R m8 , R m9 , R m10 , R m11 , R m12 , R m13 , R m14 and R m15 are independently selected
  • Y is O
  • degradable linking group has the formula: wherein R 5 , R 6 , R 7 and R 8 are as defined above:
  • Y is C(R p R q ).
  • the chemical formula of such a degradable linking group is as follows, wherein X, U 1 , U 2 , U 3 , U 4 , U 5 and U 6 are as defined above:
  • the degradable Such linking groups include, but are not limited to the following examples, wherein X, R 5, R 6, R 7 and R 8 are as defined above:
  • R 9 is selected from the group consisting of hydrogen atom, lower alkyl group, lower alkoxy group, acyl group, acyloxy group, alkoxycarbonyl group, aryl group, phenyl group, benzyl group, halogen, haloformyl group, halogenated lower alkyl group, trifluoro Methyl, trifluoromethoxy, trichloromethyl, cyano, isocyanide, isocyanate (or salt), isothiocyanate, thiocyanate (or salt), lower alkylthio, amino, imino , amino lower alkyl, lower alkylamino, lower alkylamino, hydroxy, hydroxyalkyl, nitro, nitrile, isonitrile, pyridyl, azide, carboxyl, carboxamido, acetate, sulfanyl, carbonate Or salt), carbamate, lower alkylcarbamoyl, bis lower alkylcarb
  • R 9 is selected from the group consisting of -SO 2 -OH, -SO 2 -NR m1 R m2 , -SO 2 -R m3 , -OR m4 , -SR m5 , -NR m6 R m7 , -C(O)R m8 ,- C(O)OR m9 , -OC(O)R m10 , -NHC(O)R m11 , -C(O)NR m12 R m13 , -NHC(O)NR m14 R m15 , where R m1 , R m2 , R m3 , R m4 , R m5 , R m6 , R m7 , R m8 , R m9 , R m10 , R m11 , R m12 , R m13 , R m14 and R m15 are independently selected from a hydrogen atom (H
  • R 5 , R 6 , R 7 , R 8 , and R 9 comprise a functional group such as a hydroxyl group, an amino group, a carboxyl group, a thiol group, and the like, and may form a covalent bond with a crosslinking functional group of the reactive polymer.
  • R 5 , R 6 , R 7 , R 8 , and R 9 comprise an azide or alkynyl group, which can be reacted by a click chemistry with a corresponding alkynyl or azide on the reactive polymer.
  • R p and R q are a hydrogen atom (H).
  • a group of degradable linking groups in this class have the following chemical formula. Wherein X, R 5 , R 6 , R 7 and R 8 are as defined above:
  • the chemical formula of the set of degradable linking groups is as follows:
  • such a degradable linking group has the formula: wherein X, R 6 and R 7 are as defined above:
  • the group introduces a formula of the formula wherein X, R 6 and R 7 are as defined above:
  • X is OH or HN-R 0 in the above formula.
  • X is OH in the above formula.
  • X is HN-R 0 in the above formula.
  • X is NH 2 in the above formula.
  • this group of degradable linking groups include:
  • Y 1 is C(R 3 R 4 ).
  • a class of degradable linking groups containing a 6-membered ring has the following chemical formula:
  • X is selected from -OH or -HN-R 0 ;
  • Y is selected from:
  • such degradable linking groups include, but are not limited to, the following examples, wherein X, Y, and R 3 - R 9 are as defined above:
  • Y is selected from
  • Y is C(R p R q ), and the formula of such a degradable linking group is as follows, wherein X, R 3 -R 9 , R p and R q are as defined above:
  • Examples of the group of degradable linking groups include, but are not limited to, the following chemical formula:
  • Y is CH 2
  • the chemical formula of such a degradable linking group is as follows, wherein X and R 5 -R 8 are as defined above:
  • such degradable linking groups have the formula: wherein R 5 -R 8 are as defined above:
  • X is OH or HN-R 0.
  • X is OH
  • X is HN-R 0 .
  • Examples of the group of degradable linking groups include, but are not limited to, the following chemical formula:
  • U 5 is C
  • Y 1 is NR n
  • Y is C(R p R q )
  • a class of degradable linking groups comprising a 6-membered ring has the formula: U 1 -U 4 , U 6 , R n , R p , R q and X are as defined above:
  • such degradable linking groups include, but are not limited to, the following formula, wherein R 5 -R 8 , R n , R p , R q , and X are as defined above:
  • U 5 is C
  • Y 1 is O
  • Y is CR p R q
  • a class of degradable linking groups comprising a 6-membered ring has the formula: U 1 -U 4 , U 6 ,R p , R q and X are defined as above:
  • such degradable linking groups include, but are not limited to, the formula wherein R 5 -R 8 , R p , R q , and X are as defined above:
  • Y 1 is C(O), C(S), SO or SO 2
  • chemical formula of the group of degradable linking groups is as follows:
  • X is selected from -OH or -HN-R 0 ;
  • Y is selected from:
  • R O , R 0 , R p , R q and U 1 -U 6 are as defined above.
  • the group of degradable linking groups has the formula: wherein R 5 -R 8 are as defined above:
  • Y 1 is C(O), and the chemical group of the group of degradable linking groups is as follows, wherein R 5 -R 8 are as defined above:
  • Y is NR O , wherein R O is as defined above.
  • Y is CR p R q , wherein R p and R q are as defined above.
  • X is OH or HN-R 0 , wherein R 0 is as defined above.
  • J 1 is C(R 10 R 11 ), and a class of degradable linking groups comprising a 6-membered ring has the following chemical formula:
  • U 1 , U 2 , U 3 , U 4 , U 5 , U 6 are independently selected from CR 12 and N;
  • X is selected from -OH or -HN-R 0 ;
  • Y is selected from:
  • R 0 , Ro, R p , R q and R 10 - R 12 have the same meanings as defined above.
  • Y is selected from the group consisting of NR O and O (provided X is not OH).
  • Y is C(R p R q ).
  • such an introduction group has the formula e, wherein R 5 -R 8 , R p , R q , R 10 and R 11 are as defined above:
  • R 10 , R 11 , R p , R q are a hydrogen atom (H), and the chemical group of such an introduction group is as follows, wherein R 5 -R 8 are as defined above:
  • R 10 , R 11 , R p , R q are a hydrogen atom (H), and the chemical group of such an introduction group is as follows, wherein R 5 -R 8 are as defined above:
  • X is OH or HN-R 0 , wherein R 0 is as defined above.
  • X is OH
  • X is HN-R 0 wherein R 0 is as defined above.
  • X is NH 2 .
  • degradable linking groups include, but are not limited to, the following chemical formula:
  • a class of degradable linking groups including a five-membered ring has the following chemical formula:
  • Z 1 , Z 2 , Z 3 , Z 4 , Z 5 are independently selected from the group consisting of CR 12 , N, NR 13 , O and S;
  • X is OH or HN-R 0 ;
  • Y is selected from:
  • Y 1 is selected from
  • J 1 is C(R 10 R 11 ) or a covalent bond
  • R 13 is selected from the group consisting of a hydrogen atom, a lower alkyl group, a lower alkoxy group, an acyl group, an acyloxy group, an alkoxycarbonyl group, an aryl group, a phenyl group, a benzyl group, a halogen group, a halogenoformyl group, a halogenated lower alkyl group, and a trifluoro group.
  • R 13 is selected from -SO 2 -OH, -SO 2 -NR m1 R m2 , -SO 2 -R m3 , -C(O)R m8 , -C(O)OR m9 , -C(O)NR m12 R m13 , wherein R m1 , R m2 , R m3 , R m8 , R m9 , R m12 , R m13 , independently selected from a hydrogen atom (H), a (C 1 -C 18 ) alkyl group, an aryl group, (C 1 -C 18 alkyl)OH, (C 1 -C 18 alkyl) SH, (C 1 -C 18 alkyl)COOH, (C 1 -C 18 alkyl)NH 2 , (C 0 -C 4 alkyl (C 5 -C 6 cycloalkyl), (C 0 -C 10 alkyl) (C 5
  • R 13 comprises a functional group such as a hydroxyl group, an amino group, a carboxyl group, a thiol group, and the like, and can form a covalent bond with a crosslinking functional group of the reactive polymer.
  • R 13 comprises an azide or alkynyl group and can be reacted by a click chemistry with a corresponding alkynyl or azide on the reactive polymer.
  • R 0 , Ro, R p , R q , R 3 , R 4 , and R 10 - R 12 are as defined above.
  • Y 1 is a covalent bond
  • J 1 is C(R 10 R 11 )
  • a class of degradable linking groups including a five-membered ring has the following chemical formula:
  • J 1 is C(R 10 R 11 ), Y 1 is a covalent bond, Y is C(R p R q ), and a class of degradable linking groups including a five-membered ring has the following chemical formula:
  • Y is C(R p R q )
  • J 1 is C(R 10 R 11 )
  • a class of triazole-based degradable linking groups has the following chemical formula:
  • X is OH or HN-R 0 ;
  • X is OH
  • X is HN-R 0 ;
  • X is NH 2 .
  • a class of triazole-based degradable linking groups has the formula: wherein R 5 is as defined above:
  • Y is C(R p R q ), J 1 is C(R 10 R 11 ), and a triazole-based degradable linking group has the formula: wherein R 5 is as defined above:
  • X is OH or HN-R 0 ;
  • X is OH
  • X is HN-R 0 ;
  • X is NH 2 .
  • Y is C(R p R q )
  • J 1 is C(R 10 R 11 )
  • a class of tetrazole-based introduction groups are as follows, wherein all variables are as defined above:
  • X is OH or HN-R 0 ;
  • X is OH
  • X is HN-R 0 ;
  • X is NH 2 .
  • Y is C(R p R q ), J 1 is C(R 10 R 11 ), and a class of imidazole-based degradable linking groups has the formula: wherein R 5 and R 6 are defined Same as above:
  • imidazole-based degradable linking groups include, but are not limited to, the following chemical formula:
  • n16 and m17 are each an integer of 1-10.
  • X is OH or HN-R 0 ;
  • X is OH
  • X is HN-R 0 ;
  • X is NH 2 .
  • Y is C(R p R q ), J 1 is C(R 10 R 11 ), and a pyrrole-based degradable linking group has the formula: wherein R 5 -R 7 are defined Same as above:
  • X is OH or HN-R 0 ;
  • X is OH
  • X is HN-R 0 ;
  • X is NH 2 .
  • Y is C(R p R q ), J 1 is C(R 10 R 11 ), and a pyrazole-based degradable linking group has the formula: wherein R 5 and R 6 The definition is the same as above:
  • X is OH or HN-R 0 ;
  • X is OH
  • X is HN-R 0 ;
  • X is NH 2 .
  • each of R 5 and R 6 is a hydrogen atom.
  • Y 1 and J 1 are covalent bonds
  • a class of degradable linking groups has the following chemical formula:
  • Y is C(R p R q ).
  • a class of triazole-based degradable linking groups has the formula: wherein R 5 is as defined above:
  • a class of triazole-based degradable linking groups has the formula: wherein R 5 is as defined above:
  • a class of triazole-based degradable linking groups has the formula: wherein R 5 is as defined above:
  • a class of tetrazole-based degradable linking groups have the following chemical formula:
  • a class of imidazole-based degradable linking groups has the formula: wherein R 5 and R 6 are as defined above:
  • a pyrrole-based degradable linking group has the formula: wherein R 5 -R 7 are as defined above:
  • a pyrazole-based degradable linking group has the formula: wherein R 5 and R 6 are as defined above:
  • X is OH or HN-R 0 ;
  • X is OH
  • X is HN-R 0 ;
  • X is NH 2 .
  • Y 1 is C(R 3 R 4 ), and the chemical formula of a class of degradable linking groups is as follows:
  • Y is C(R p R q ).
  • a class of triazole-based degradable linking groups has the formula: wherein R 5 is as defined above:
  • a class of triazole-based degradable linking groups has the formula: wherein R 5 is as defined above:
  • a class of triazole-based degradable linking groups have the following chemical formula, wherein R 5 is as defined above:
  • a class of tetrazole-based degradable linking groups have the following chemical formula:
  • a class of imidazole-based degradable linking groups has the formula: wherein R 5 and R 6 are as defined above:
  • a pyrrole-based degradable linking group has the formula: wherein R 6 and R 7 are as defined above:
  • a pyrazole-based degradable linking group has the formula: wherein R 5 and R 6 are as defined above:
  • X is OH or HN-R 0 ;
  • X is OH
  • X is HN-R 0 ;
  • X is NH 2 .
  • Y 1 is C(O)
  • a class of degradable linking groups has the following chemical formula:
  • X is OH or HN-R 0 ;
  • Y is selected from:
  • A, B, Ro, R 0 , R p and R q are as defined above.
  • a and b are as defined above, and m and n are each an integer independently selected from 0, 1, 2, 3, 4, 5, and 6.
  • the degradable linker such as the following chemical formula, wherein R 5 and R 6 are as defined above:
  • Y is C(R p R q ), and the chemical formula of such a degradable linking group is as follows, wherein R 5 and R 6 are as defined above:
  • X, Y and C(O) constitute a natural or unnatural amino acid or hydroxy acid residue.
  • Non-limiting examples include alanine, arginine, asparagine, aspartic acid, cysteine, glycine, glutamic acid, Histidine, isoleucine, leucine, lysine, methionine, valine, glutamine, phenylalanine, serine, threonine, proline, tryptophan, casein Amino acid, aminoisobutyric acid, sarcosine, glycolic acid and phenyllactate.
  • degradable linking groups include, but are not limited to:
  • R 5 and R 6 are as defined above.
  • degradable linking groups include, but are not limited to:
  • X is OH or HN-R 0 ;
  • X is OH
  • X is HN-R 0 ;
  • X is NH 2 .
  • two R groups attached to the same atom or to two atoms separated by a bond (selected from R 0 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R O , R n , R p , R q ) and the atom to which they are bonded constitute a C 4 -C 10 cycloalkyl group, a cycloolefin a saturated or unsaturated monocyclic, polycyclic, and fused ring, optionally substituted with at least one group other than a hydrogen atom (H); a saturated or unsaturated monoheterocyclic ring, a heterocyclic ring, and a thick a heterocyclic ring, optionally substituted with at least one group other than a hydrogen atom (H); or two R groups together with an atom to which they are attached constitute a single aromatic or polyaromatic ring,
  • At least one group is selected from the group consisting of lower alkyl, lower alkoxy, acyl, acyloxy, alkoxycarbonyl, aryl, phenyl, benzyl, halogen, haloformyl, halo lower alkyl, three Fluoromethyl, trifluoromethoxy, trichloromethyl, cyano, isocyanide, isocyanate (or salt), isothiocyanate, thiocyanate (or salt), lower alkylthio, amino, sub Amino, amino lower alkyl, lower alkylamino, lower alkylamino, hydroxy, hydroxyalkyl, nitro, nitrile, isonitrile, pyridyl, azide, carboxyl, carboxamido, acetate, sulfanyl, carbonate (or salt), carbamate, lower alkylcarbamoyl, bis lower alkylcarbamoyl, sulfonate,
  • At least one of R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R p , R q may form with a reactive polymer.
  • the hydrogel can consist of a backbone moiety and an optional crosslink.
  • the skeleton portion is characterized by having a branched core extending from the core by at least 2 chains, preferably 2-16, more preferably 4-8.
  • the branched core may be composed of a combined form of a polyhydric or lower alcohol, preferably pentaerythritol, three quarters Alcohol, hexaglycerin, sorbitol, mannitol, polyvinyl alcohol, trimethylolpropane, sucrose, fructose, glucose, dextran, cellulose, starch, amylose, hyaluronic acid, or
  • the branched core may be composed of a combined form of a polyvalent or a low-amine amine, such as ornithine, diaminobutyric acid, trilysine, tetralysine, pentalysine, hexa-lysine, hepta-lysine. , eight lysine, nine lysine, ten lysine or
  • the chain extending from the branched core can be of any length.
  • the chain atoms are all carbon atoms.
  • the chain atoms of the backbone are selected from the group consisting of C, O, N, and S, and the chain atoms can be selected according to their expected solubility to provide a more soluble chain.
  • the chain comprises an optional linking group that is degradable by an enzyme or that is automatically cleaved under in vivo conditions.
  • the chain consists of several fragments joined together by an ester bond or an amide bond.
  • Chains can include, but are not limited to, PEG, long chain fatty acids, natural or unnatural amino acids (eg, beta-alanine, gamma-aminobutyric acid, gamma-glutamic acid), short peptides (eg, beta-alanine-beta) - alanine, ⁇ -glutamic acid- ⁇ -glutamic acid), or a combination of two or more of the above (for example, optional long-chain fatty acids, PEG, amino acids, short peptides, etc. are linked by covalent bonds Long chain formed together).
  • the strand is a peptide of any length.
  • Exemplary chains are from about 1 to 50 amino acids in length, from 5 to 50, from 3 to 5, from 5 to 10, or from 5 to 15 amino acids in length.
  • the chain is polylysine, polyglutamic acid, polyaspartic acid, a copolymer of these amino acids, and a mixed polymer of these amino acids with other amino acids, such as serine.
  • PEG-based polymeric chains are PEG-based polymeric chains.
  • the PEG-based polymeric chain can be linear, branched or branched.
  • the PEG-based polymeric chain is attached to the core at one end and the other end to a structure having at least one crosslinking functional group and at least one optional reactive functional group.
  • such a structure has a hyperbranched portion.
  • the structure of the polymeric chain may be the same or different.
  • the number of PEG fragments on each polymeric chain can vary.
  • the PEG-based polymeric chain may contain an alkyl group, an aryl group, and a hetero atom in between.
  • the hyperbranched portion can provide more functional groups.
  • additional functional groups of the hyperbranched moiety can increase the amount and density of crosslinks.
  • additional functional groups of the hyperbranched moiety can increase the loading of the drug molecule.
  • a preferred structure for the PEG-based polymeric chain from which the branched core extends is a multi-arm PEG derivative such as the 4-arm and 8-arm PEG derivatives provided by commercial companies.
  • the sum of the cross-linking functional group and the reactive functional group of the backbone moiety is equally divided by the number of polymeric chains of the PEG-based group extending from the branched core such that the same number of cross-links are present on the polymeric chain of each PEG-based group.
  • Functional groups and reactive functional groups are equally divided by the number of polymeric chains of the PEG-based group extending from the branched core such that the same number of cross-links are present on the polymeric chain of each PEG-based group.
  • the hydrogel backbone of the present invention has the general formula J(L 1 -B 1 ) n1 , wherein J is a branched core; L 1 is a chain extending therefrom, and the two ends are respectively The price key connects J and B 1 .
  • L 1 may be a linear, branched or branched structure.
  • the chain atoms are all carbon atoms.
  • the chain atoms in the main chain L 1 is selected from C, O, N and S, the chain atoms may be selected according to their expected solubility, solubility in order to provide a more suitable chain.
  • L 1 comprises an optional linking group that is degradable by an enzyme or that is automatically cleaved under in vivo conditions.
  • L 1 may include, but is not limited to, PEG, long chain fatty acids, natural or unnatural amino acids (eg, ⁇ -alanine, ⁇ -aminobutyric acid, ⁇ -glutamic acid), short peptides (eg, ⁇ -alanine- --alanine, ⁇ -glutamic acid- ⁇ -glutamic acid), or a combination of two or more of the above (for example, an optional long-chain fatty acid, PEG, amino acid, short peptide, etc. through a covalent bond Long chains formed together.
  • L 1 is a PEG-based polymeric chain. More preferably, each L 1 is independently selected from the group -(CH 2 ) n4 (OCH 2 CH 2 ) n L n5 or -(CH 2 ) n4 (CH 2 CH 2 O) n L n5 , wherein n4 is 0- An integer of 5, n is an integer from 1 to 1000, and L n5 is a bond or a chemical functional group linking the L 1 terminal and B 1 . In certain embodiments, L 1 is a peptide of any length.
  • L 1 is polylysine, polyglutamic acid, polyaspartic acid, a copolymer of these amino acids, and a mixed polymer of these amino acids with other amino acids, such as serine;
  • B 1 is A structure having at least one crosslinking functional group C 2 and at least one optional reactive functional group C 1 .
  • the reaction functional group and the crosslinking functional group may be the same or different. Functional groups on the same skeleton cannot react with each other.
  • n 1 is an integer from 2-16.
  • Each of L 1 and B 1 can be independently selected.
  • B 1 has a dendrimer structure.
  • L 1 and B 1 are linked by an amide bond.
  • the structure of a 4-arm hydrogel backbone is as follows:
  • C 1 is a reactive functional group
  • C 2 is a cross-linking functional group.
  • C 2 with the crosslinking functional group crosslinked orthogonal reaction, C 1 reaction does not crosslinked with a crosslinking functional group.
  • the hydrogel backbone has the formula (C 1 -L 1 ) n2 J(L 1 -C 2 ) n3 , wherein J is a branched core; L 1 is a chain extending therefrom; C 1 is a reactive functional group and C 2 is a crosslinking functional group.
  • the reactive functional group and the cross-linking functional group may be the same or different, and the functional groups on the same skeleton may not react with each other; n2 and n3 are integers of 0-16.
  • the hydrogel skeleton of the present invention has the formula (C 1 -L 1 ) 4 J(L 1 -C 2 ) 4 , wherein J is a branched core such as hexaglycerol and tripentaerythritol; L 1 It is a chain extending from it; C 1 is a reactive functional group; and C 2 is a cross-linking functional group.
  • L 1 is a PEG-based polymeric chain.
  • each L 1 is independently selected from the group -(CH 2 ) n4 (OCH 2 CH 2 ) n L n5 or -(CH 2 ) n4 (CH 2 CH 2 O) n L n5 , wherein n4 is 0- An integer of 5, n is an integer from 1 to 1000, and L n5 is a bond or a chemical functional group linking the L 1 terminal and C 1 , C 2 .
  • L 1 and C 1 , C 2 are linked by an amide bond.
  • C 1 and C 2 of each skeleton portion (C 1 - L 1 ) 4 J(L 1 - C 2 ) 4 can be independently selected.
  • the structure of the 8-arm hydrogel backbone is as follows:
  • C 1 and C 2 may be attached to a bifurcated structure or a hyperbranched compound, such as B 1 described below.
  • B 1 is a highly branched polypeptide.
  • the highly branched polypeptide comprises one or more lysines.
  • Lysine can be either L-form or D-form. D-lysine is better able to resist enzymatic hydrolysis.
  • J is the core with branches.
  • the number of lysine residues per branch can be increased or decreased as needed, part of the amino group of lysine can be used for crosslinking, and part can be used for loading bioactive substances.
  • the backbone moiety has a molecular weight ranging from 1 to 40 kDa, more preferably from 5 to 20 kDa.
  • a cross-linker is a compound comprising at least two cross-linking functional groups and at least one optional biodegradable linking group.
  • the cross-linking functional group is linked to the cross-linker by a biodegradable linking group, and a chain for attachment can be inserted between the cross-linking functional group and the biodegradable linking group as needed.
  • the cross-linking functional group of the cross-linker is capable of reacting with a cross-linking functional group of the hydrogel backbone to form a covalent bond.
  • the functional groups of the cross-linking materials may be the same or different (heterobifunctional).
  • the first functional group of the heterobifunctional crosslinker reacts with the cross-linking functional group of the hydrogel backbone, and the second functional group reacts with the cross-linking functional group of the same or the second hydrogel backbone .
  • the crosslinker has more than one biodegradable linking group. These biodegradable linking groups may be the same or different. Functional groups on the same cross-linker cannot react with each other.
  • the structure of the crosslinked product may be a chain type such as C 4 -Z 2 -L 2 -C 4 , C 4 -Z 2 -L 2 -Z 2 -C 4 , C 4 -Z 2 -L 2 -C 4 ' , C 4 -Z 2 -L 2 -Z 2 -C 4 ' or C 4 -Z 2 -L 2 -Z 2 '-C 4 ', wherein L 2 is a chain connecting the two ends, and C 4 and C 4 ' are Crosslinking functional groups, Z 2 and Z 2 ' are optional biodegradable linking groups.
  • a chain for attachment can be inserted between C 4 and Z 2 , C 4 ' and Z 2 ' as needed.
  • the structure of the crosslinked product may be a multi-arm structure, such as a 4-arm structure (cross type), an eight-arm structure, etc., and the general formula is O(L 2 -Z 2 -C 4 ) n6 , where O is a branched core, L 2 is a chain extending therefrom, C 4 is a cross-linking functional group, Z 2 is an optional biodegradable linking group, and n6 is an integer of 2-8. .
  • the Z 2 of each branch may be the same or different.
  • the C 4 of each branch may be the same or different.
  • the structure of the 4-arm cross-linker is as follows:
  • C 4 absent, Z 2 may be reacted directly with the crosslinking functional groups of the backbone portion to form a covalent bond.
  • cross-linking functional group of the hydrogel skeleton of the present invention and the cross-linking functional group of the corresponding site of the cross-linking material are associated reaction pairs.
  • association reactions for some common, non-limiting functional groups include:
  • the amino group can be reacted with a carboxylic acid, an aldehyde and a ketone.
  • Thiol groups can participate in Thiol-Ene Reactions.
  • the thiol group can be reacted with maleimide, iodoacetic acid, vinyl sulfone, vinyl amide, acrylamide, acrylate, halocarbonyl, enone.
  • the azide compound can be reacted with an alkynyl group (for example, an alkyne having 2 to 20 carbon atoms, a cyclooctyne, an alkyne), a bicyclononyne (BCN), a maleimide.
  • an alkynyl group for example, an alkyne having 2 to 20 carbon atoms, a cyclooctyne, an alkyne
  • BCN bicyclononyne
  • the azide-alkynyl cycloaddition reaction includes a copper-catalyzed reaction and a strain-promoted reaction.
  • Common functional groups of the latter include dibenzocyclooctyne (DBCO), monofluorocyclooctyne (MFCO), difluorinated cyclooctyne (DIFO), and aryl-free cyclooctyne ( Aryl-less cyclooctyne, ALO).
  • the reaction rate of different cyclooctyne functional groups with azide compounds can be 1000 times different (DBCO>MFCO>ALO).
  • the azide compound can selectively react with one or several of the cyclooctyne functional groups, for example, first with DBCO.
  • Another class that promotes the reaction with an azide compound by tension is a bicyclic decyne compound.
  • the nitrone can be reacted with an alkynyl group (e.g., cyclooctyne) or a bicyclic decyne.
  • an alkynyl group e.g., cyclooctyne
  • a bicyclic decyne e.g., cyclooctyne
  • a Diels-Alder cycloaddition reaction such as maleimide, can be reacted with furan, 1,3-diene, cyclopentadiene.
  • the tetrazine can be reacted with an olefin.
  • an olefin for example, 4-(6-methyl-1,2,4,5-tetrazine)phenylacetic acid and norbornene are inverse-electron demand Diels-Alder cycloaddition reaction (inverse-electron demand Diels-Alder cycloaddition reaction )reaction.
  • tetrazine can also react with trans-cyclooctene (TCO).
  • Alkoxyamine can be condensed with a ketone such as pyruvamide to form oxime.
  • the aldehyde can be reacted with hydroxylamine or hydrazide.
  • the tetrazole reacts with the olefin.
  • the dithioester can be reacted with a diene.
  • Bismuth can react with maleimide.
  • Any chemical reaction known in the art can be applied to cross-linking, including acylation, reductive alkylation, Michael addition, thiol alkylation or by reactive groups on one molecule (eg aldehyde, amino, ester, sulfur) Alcohol, ⁇ -haloacetyl, maleimide, N-hydroxysuccinimide ester or sulfhydryl) a reactive group for the same molecule or another molecule (eg aldehyde, amino, ester, thiol, ⁇ -)
  • the haloacetyl, maleimide, N-hydroxysuccinimide ester or sulfhydryl group is chemiselectively conjugated or linked.
  • Activating groups used in the reaction include, but are not limited to, N-hydroxysuccinimide ester, sulfone, maleimide, triflate, trifluoroethyl sulfonate, aziridine , ethylene oxide and 5-pyridyl.
  • the hydrogel backbone moiety is directly attached by a biodegradable linkage or a biodegradable linkage group, without the need for a crosslinker.
  • the backbone moieties can be joined together by a crosslinker.
  • Each crosslinker comprises at least one biodegradable linking group.
  • the biodegradable hydrogel can contain one or more different types of crosslinks.
  • the cross-linker is a compound comprising at least two cross-linking functional groups, at least one biodegradable linking group, and at least one optional reactive functional group.
  • the cross-linking functional group or reactive functional group is linked to the cross-linker via a biodegradable linking group.
  • a chain for attachment can be inserted between the cross-linking functional group or the reactive functional group and the biodegradable linking group as needed.
  • the cross-linking functional group of the cross-linker reacts with a cross-linking functional group of the hydrogel backbone to form a covalent bond.
  • the cross-linking functional groups of the cross-linking materials may be the same or different (heterobifunctional).
  • the first cross-linking functional group of the heterobifunctional cross-linker reacts with the cross-linking functional group of the hydrogel backbone, and the second cross-linking functional group cross-links with the same or another hydrogel backbone Functional group reaction. Functional groups on the same cross-linker cannot react with each other.
  • the structure of such a crosslinked product may be a chain type.
  • the cross-linking formula is B 2 -Z 2 -L 2 -Z 2 -B 2 , B 2 -Z 2 -L 2 -B 2 , B 2 -Z 2 -L 2 -Z 2 -B 2 ' or B 2 -Z 2 -L 2 -Z 2 '-B 2 ', wherein L 2 is a chain connecting the two ends, and B 2 and B 2 ' respectively have at least one crosslinking functional group and at least one Selected reaction function group.
  • B 2 or B 2 ' has an optional hyperbranched moiety such that multiple cross-linking functional groups and multiple reactive functional groups can be provided.
  • Z 2 and Z 2 ' are optional biodegradable linking groups.
  • B 2, and B 2 ', Z 2, and Z 2' may be the same or different.
  • the structure of the chain crosslinker is as follows:
  • Z 2 is an optional degradable linking group
  • C 4 is a crosslinking functional group
  • C 6 is a reactive functional group
  • One or more optional biodegradable linking groups may be included in the branched structure of B 2 or B 2 '.
  • the structure of the chain crosslinks is as follows:
  • Z 2 and Z 3 are optional degradable linking groups
  • C 4 is a crosslinking functional group
  • C 6 is a reactive functional group.
  • the crosslinker is of the formula B 2 -L 2 -B 2 , or B 2 -L 2 -B 2 ', wherein L 2 is a chain connecting the two ends, and B 2 and B 2 ' have at least A cross-linking functional group and at least one optional reactive functional group, the cross-linking functional group and the reactive functional group, may each be linked to an optional degradable linking group. Functional groups on the same cross-linker cannot react with each other.
  • the structure of the chain crosslinker is as follows:
  • the PEG-based chain crosslink structure of the branched structure (not including the optional degradable linking group and the reactive functional group or crosslinking functional group attached thereto) is as follows:
  • the cross-linker has the formula O(L 2 -Z 2 -B 2 ) n6 , wherein O is a branched core, L 2 is a chain extending therefrom, and Z 2 is optional.
  • Degradable linking group B 2 has at least one crosslinking functional group and at least one optional reactive functional group, and the reactive functional group or crosslinking functional group can be linked to an optional degradable linking group.
  • B 2 has a dendrimer structure.
  • N6 is an integer from 2-8.
  • L 2 is a PEG-based polymeric chain, and the two ends are respectively linked to a branched core and Z 2 by a covalent bond. More preferably, each L 2 is independently selected from the group consisting of formula (CH 2 ) n4 (OCH 2 CH 2 ) n L n5 or (CH 2 ) n4 (CH 2 CH 2 O) n L n5 , wherein n4 is 0-5 An integer, n is an integer from 1 to 1000, and L n5 is a bond or a chemical functional group linking the L 2 terminal and Z 2 .
  • L 2 and Z 2 are linked by an amide bond.
  • Z 2 and B 2 of each O(L 2 -Z 2 -B 2 ) 4 can be independently selected.
  • B 2 in a branched structure may comprise one or more optional biodegradable linker.
  • the structure of the 4-arm cross-linker is as follows:
  • Z 2 , Z 3 , C 4 and C 6 are as defined above. In certain embodiments, Z degradation half-lives longer than 2 Z 3.
  • the cross-linker has the formula O(L 2 -B 2 ) n6 , n 6 is an integer from 2-8, and B 2 has at least one cross-linking functional group and at least one optional reactive functional group.
  • the interconnecting functional groups and reactive functional groups can be attached to an optional degradable linking group, respectively.
  • the structure of the 4-arm cross-linker is as follows:
  • C 4 or C 6 is absent, and Z 2 , Z 3 may react directly with a functional group of the backbone moiety to form a covalent bond.
  • branch structure with B 2 (not including optional degradable linking groups and reactive functional groups attached thereto, or a crosslinking functional group) structure crosslinked multi-arm PEG groups are as follows:
  • the monomers that make up the polymeric crosslinker moiety are linked by a biodegradable linkage.
  • the cross-linking moiety include polymers based on polyglycolic acid or polylactic acid.
  • the cross-linking functional group of the four-arm hydrogel skeleton reacts with the cross-linking functional group of the four-arm cross-linker to form a network structure.
  • the hydrogel backbone and crosslinker are four arm 20 kDa PEG based.
  • a non-limiting example is as follows:
  • C 2 is a cross-linking functional group of the hydrogel skeleton, and the cross-linking functional group C 4 of the corresponding site of the cross-linker is an associated reaction pair.
  • C 1 is the reactive functional group of the hydrogel backbone and Z 2 is a biodegradable linking group.
  • the reactive functional groups contained in the backbone and crosslinks in the hydrogel can be used for a variety of purposes, such as loading biologically active substances or biomarkers. These reactive functional groups can be used directly and can be converted or derivatized for use in other orthogonal reaction functional groups.
  • the amount of hydrogel injected should be as small as possible, thus requiring a high drug loading capacity of the hydrogel. Even if the hydrogel skeleton contains a hyperbranched portion, when the drug molecule is relatively large, steric hindrance affects the loading of the skeleton.
  • the use of a branched or bifurcated structure in the crosslinks increases the number of reactive functional groups and reduces the steric hindrance by the length of the branched or bifurcated structure, thus hydrogels The drug-loading potential has increased significantly.
  • the bioactive substance in the present invention may be linked to a degradable linking group, and the degradable linking group may be coupled to the hydrogel through an optional reactive functional group, thereby loading the biologically active substance on the hydrogel.
  • the biologically active substance of the present invention may be linked to a degradable linking group by a nitrogen atom-containing structure (e.g., an amino group) or a hydroxyl group, and the linking group and the optional reactive functional group
  • the hydrogel is coupled by means of a hydrogel - an optional reactive functional group - a degradable linking group - a biologically active substance.
  • an optional reactive functional group of the four-arm hydrogel backbone is linked to the biologically active material via a biodegradable linking group.
  • a biodegradable linking group is as follows:
  • C 2 is a cross-linking functional group of the hydrogel skeleton, and the cross-linking functional group C 4 of the corresponding site of the cross-linker is an associated reaction pair.
  • C 1 is a reactive functional group of the hydrogel skeleton and is associated with the reactive functional group C 3 .
  • Z 1 and Z 2 are optional biodegradable linking groups. Drug stands for biologically active substances. In certain embodiments, C 3 absent, Z 1 -drug directly with C 1 to covalent bonds.
  • the release (>90%) of the majority of the biologically active material preferably occurs before a significant amount of the backbone moiety (<10%) has been released.
  • This can be achieved by inserting a cross-linker between the backbones and a linker moiety between the hydrogel and the biologically active substance to insert a different half-life degradable linking group.
  • the degradable linking group for the crosslinks between the backbones has a longer half life.
  • the four cross-linking functional groups of the eight-arm hydrogel skeleton react with the four cross-linking functional groups of the four-arm cross-linker to form a network structure.
  • the hydrogel backbone is an eight-arm 40 kDa PEG-based and the cross-linker is a four-arm 20 kDa PEG-based.
  • a non-limiting example is as follows:
  • the remaining four eight-arm reaction hydrogel matrix functional groups C 1 and degrading the biologically active substance is connected via biodegradable linker.
  • C 1 , C 2 , C 3 , C 4 , Z 1 , Z 2 and Drug are as defined above.
  • the hydrogel of the present invention is hydrolyzed in the body, and may be hydrolyzable by hydrolysis of a degradable bond in the hydrogel skeleton, or may be hydrolyzed by a degradable bond in the crosslinked product, or hydrolyzed by both the hydrogel skeleton and the crosslinked product. .
  • the degradable linking group of the present invention bonds the two hydrogel backbone units or between the hydrogel backbone and the crosslinker with an amide bond.
  • the amide bond is the most stable chemical bond in the living body, and the hydrogel skeleton or cross-linker constructed on the basis of polyethylene glycol, polypropylene glycol or the like does not contain a typical cleavage site of the enzyme in the human body. Therefore, the hydrogel constructed in this manner in the present invention has good in vivo stability, and the rate of degradation is determined by the degradable group. According to the inventors, this is the first system to construct a hydrogel with an amide bond and to automate the cleavage of the amide bond to achieve hydrogel degradation.
  • Hydrogels using conventional non-covalent encapsulation methods typically degrade the hydrogel by hydrolysis of the cross-linked ester linkages, releasing the bioactive material.
  • Hydrogels using the covalent reservoir method are capable of degradation, typically using a large number of ester linkages in the backbone, and biologically active materials are often attached to the hydrogel via ester linkages.
  • Rate of hydrolysis of ester bonds for polymers The rate of change is narrower, and the network chemistry and structure of the hydrogel play a greater role in the degradation rate of the hydrogel. Therefore, the delivery system for each bioactive substance needs to be optimized separately.
  • the hydrogel of the present invention has the advantage of using a degradable linking group under physiological conditions that does not require enzyme catalysis.
  • the hydrogel backbone of the present invention may comprise a degradable linking group internally, or the hydrogel backbones may be linked by a degradable linking group, or the hydrogel backbone may be linked by a crosslinker comprising a degradable linking group. .
  • the network chemistry and structure of the hydrogel are basically unchanged, so that the release rate of the bioactive substance and the rate of degradation of the hydrogel depend on the breaking kinetics of the degradable linking group, and the appropriate degradation rate can be selected according to the requirements of the drug. It is sufficient to degrade the linking group.
  • the half-life of the degradable linking group provided by the present invention has a wide range of variation to meet different clinical needs. These hydrogel systems do not substantially require separate optimization of the polymer structure for each bioactive material.
  • the properties of the carrier material such as biocompatibility (minimum irritancy, immunogenicity, toxicity, etc.), can be independently optimized in addition to the bioactive substance release properties.
  • Certain embodiments of the invention use a non-covalent packaging method.
  • the average pore size in the three-dimensional network of hydrogels is less than the size of the bioactive material, such as the drug encapsulated within the hydrogel, without chemical bonds to the hydrogel.
  • forming a hydrogel in the presence of a biologically active material can encapsulate the biologically active material in a gel.
  • Certain embodiments of the invention use a covalent reservoir approach. If the molecular weight of the loaded bioactive substance is from 1 to 100 Kda, the distance between the hydrogel nodes is not less than 7 nm in order to allow the bioactive substance to pass through the pores of the hydrogel. The distance between the two multi-arm backbones of the cross-linked PEG group and the core of the cross-linker is generally not shorter than the length of the 10 kDa PEG chain. If the loaded bioactive material is a small molecule compound, the distance between the hydrogel nodes is shortened, and a smaller molecular weight PEG-based multi-arm skeleton and cross-linker can be used.
  • the hydrogel of the present invention may provide a single biologically active substance, and may also provide a composition of more than one biologically active substance.
  • the bioactive substances in the composition can be used to treat the same disease and can also be used to treat different diseases.
  • the release rates of different biologically active substances may be the same or different by selecting the same or different half-life degradable linking groups.
  • the half life of the hydrogel is from 1 to 1000 hours, preferably from 1 to 5000 hours, more preferably from 1 to 1000 hours.
  • the range given in the present application, for example, 1-1000 hours, should be considered to be specifically and explicitly disclosed in the interval between them, so it is not necessary to recite a long string of values. This application includes any random range within the scope, such as 1-1000 including 1-500 and 100-500.
  • a common method of extending the duration of action of a drug in vivo is to link the polypeptide or protein to a natural or synthetic macromolecule via a hydrolyzable or non-hydrolyzable bond.
  • Biomacromolecules include albumin, polysaccharides (such as dextran), antibodies (such as IgG or IgG Fc), and the like.
  • Synthetic macromolecules include polyethylene glycol and the like.
  • the drug and the macromolecule are combined by a hydrolyzable bond (such as an ester, a carbonic acid, a hydrolyzable carbamate), and the currently used hydrolyzable bond is poor in stability, and is easily degraded or spontaneously hydrolyzed in plasma, thereby making drug release difficult to predict.
  • a hydrolyzable bond such as an ester, a carbonic acid, a hydrolyzable carbamate
  • many biologically active substances, especially those with a relatively small molecular weight lack suitable functional groups that can be conjugated to macromolecules. Both the enzymatic or fusion protein method can only reduce enzyme degradation and renal clearance to a certain extent.
  • PEG40K is the largest molecular weight polyethylene glycol currently used in clinical practice, but even if the molecular weight of polyethylene glycol is increased to 60K or 80K, the prolongation time is limited, and the molecular weight of polyethylene glycol is too large, which increases the risk of side effects. So long-term methods that rely on macromolecules have an insurmountable time limit.
  • the hydrogel of the present invention uses the subcutaneous tissue as a drug storage reservoir, and there is no problem of enzymatic degradation and renal clearance before drug release, so the administration time can be extended to weeks, months or even years.
  • the existing long-acting drugs are administered in a plurality of doses, even for multiple days, and the initial blood concentration is high, which increases the side effects of drugs with low therapeutic index.
  • bioactive prodrugs is a solution, many bioactive substances have no functional sites for conjugation, and macromolecules are conjugated to distal functional groups, which may not inhibit their activity. .
  • the hydrogel of the present invention limits the biologically active substance to the injection site before it is released, and cannot reach the target, so that physiological activity is not exhibited, and the effect is similar to the prodrug.
  • the hydrogels of the present invention can accommodate various functional group types and locations of biologically active materials.
  • the hydrogel slowly releases the drug, and the blood drug concentration curve is stable, which improves the safety of the drug with a low therapeutic index and improves the therapeutic effect of the drug.
  • protein drugs usually contain multiple cysteines or lysines, and it is difficult to conjugate at a fixed point, and a single product is often not available.
  • Most PEGylated protein drugs have this problem.
  • possible attachment sites for polyethylene glycol include lysine, serine, tyrosine, and histidine.
  • Porcine uricase is a tetramer, and each subunit contains 28-29 lysines which can theoretically react with polyethylene glycol. In fact, each subunit can be conjugated to an average of 10-11 PEG10KDa.
  • Polyethylene glycol uricase Each subunit of uric acidase in pegloticase contains an average of 9 polyethylene glycol molecules. The eight lysines of INF- ⁇ 2a can react with polyethylene glycol to form eight different products, the highest activity of which is three times the activity of the least active product (Foser et al, protein expression and purification 2003, 30 , 78-87).
  • the non-covalent encapsulation method of the present invention does not involve the reaction with a drug, the original sequence and structure of the drug in and after the hydrogel.
  • the covalent reservoir method of the present invention is linked to a drug by a covalent bond, and the attachment site may not be the same, but the original sequence and structure are still released after the drug is released, ensuring the singularity of the actual effective drug.
  • the present invention provides a method for preparing a biodegradable hydrogel, the method comprising reacting a hydrogel backbone with a crosslinker, wherein the crosslinker comprises a functional group and a biodegradable linker that can react with a corresponding site of the backbone group.
  • the biodegradable hydrogel is prepared by reacting the same backbone with a degradable crosslink.
  • the backbone is multivalent and thus nodes can be formed in the three dimensional hydrogel matrix.
  • An example is the reaction of a multi-arm PEG with a crosslinker to form a hydrogel.
  • Multi-arm PEG reagents having different reactive functional groups and molecular weights are commercially available.
  • a linear polymer is reacted with a crosslink to form a hydrogel. Examples of linear polymers include hyaluronic acid, polyvinyl alcohol, carboxymethyl cellulose, poly(2-hydroxyethyl methacrylate), dextran, chitosan, collagen, alginate, and agarose. .
  • the present invention provides methods for producing biodegradable hydrogels by reacting more than one different backbone and a degradable crosslink.
  • the degradable crosslinker comprises a functional group that reacts with the first backbone, a functional group 2 that reacts with the second backbone, and a degradable linking group.
  • Functional groups 1 and 2 can be the same or different.
  • the backbone comprises at least 2 arms, more preferably at least 4 arms.
  • the reactive polymer used in the present invention may be homopolymerized or copolymerized polyethylene glycol, polypropylene glycol, poly(N-vinylpyrrolidone), polymethacrylate, polyphosphazene, polylactide, polyacrylamide, Polyglycolic acid, polyethyleneimine, agarose, dextran, gelatin, collagen, polylysine, chitosan, alginate, hyaluronic acid, pectin, carrageenan or polyamino acid, It has a suitable reactive functional group and a cross-linking functional group in a natural state, or is derivatized to have a suitable reactive functional group and a cross-linking functional group.
  • Suitable reactive functional groups and crosslinking functional groups include, but are not limited to, amines, alcohols, thiols, carboxylic acids, maleimides, acrylates, acrylamides, azides, alkynes Classes (including cycloalkynes), 1,3-dienes (including cyclopentadienes and furans), ⁇ -halocarbonyls, N-hydroxysuccinimides, N-hydroxysulphonium Imide esters or carbonates.
  • the hydrogels of the present invention may also contain reactive functional groups that are not consumed during the gelation process, and these functional groups do not participate in the gelation process because of excessive or orthogonal reactivity. These functional groups can be used to further modify the hydrogel, for example by covalent bonding of biologically active substances.
  • the reactive functional group on the backbone is first reacted with a reactive functional group-optionally degradable linking group-bioactive material to provide an intermediate drug-loaded polymer.
  • the cross-linking functional group on the backbone then reacts with the cross-linker to form a drug-loaded degradable hydrogel.
  • the cross-linking functional group on the backbone first reacts with the cross-linker to form a hydrogel, and then the reactive functional group on the backbone reacts with the reactive functional group-optionally degradable linking group-bioactive material, A drug-loaded degradable hydrogel is formed.
  • the backbone, cross-linker, and reactive functional group-optionally degradable linking group-bioactive material are combined in a single step (one-pot method). If there are still unreacted functional groups after the drug-loaded hydrogel is formed, the excess functional group can be capped with a suitable reagent.
  • Microporous, mesoporous or macroporous hydrogels can be prepared by selecting backbones and crosslinks of different structures and sizes.
  • Microporous hydrogel refers to a hydrogel having pores having a diameter of less than 1 nm
  • a mesoporous hydrogel is a hydrogel having pores having a diameter of 1-100 nm
  • a macroporous hydrogel is water having pores having a diameter of more than 100 nm. gel.
  • the hydrogel of the present invention can be prepared in vitro and then implanted into the body.
  • the hydrogel can be made into a specific shape.
  • the hydrogel is a shaped article such as a stent or mesh.
  • the hydrogel of the present invention can be obtained by a production method in the form of fine particles.
  • the hydrogel is beaded microparticles which can be injected subcutaneously or intramuscularly by syringe.
  • the beaded particles have a diameter of from 1 to 500 microns.
  • the microparticles are 10-100 microns in diameter, preferably 20-100 microns, and most preferably 30-70 microns.
  • the microparticles can be injected using an injection needle having an inner diameter of less than 0.6 mm, preferably an injection needle having an inner diameter of less than 0.3 mm, more preferably an injection needle having an inner diameter of less than 0.25 mm, even more preferably an injection needle having an inner diameter of less than 0.175 mm, preferably an inner diameter. Injection needles smaller than 0.16 mm.
  • the hydrogel can be formed by in situ gelation.
  • the pharmaceutically acceptable hydrogel formulation ingredients are prepared first, the ingredients are mixed, and then injected or applied prior to gelation.
  • the injection can be subcutaneous, intramuscular, intraocular, intratumoral or intravenous.
  • the hydrogel of the present invention can be applied topically, for example, by using a mixed ingredient to the skin or surgical wound and then gelating it in situ.
  • Still other applications of the hydrogels of the present invention are, for example, regenerative medicine, orthopedic or orthopedic implants, medical device coatings or surgical dressings.
  • Bom benzyloxymethyl: benzyloxymethyl; Br-Z, 2-bromobenzyloxycarbonyl: 2-bromobenzyloxycarbonyl; tBu, t-butyl: tert-butyl; Bz, benzoyl: benzoyl; Bzl, benzyl: benzyl; : tert-butoxycarbonyl; CHO formyl: formyl; cHx, cyclohexyl: cyclohexyl; Cbz or Z benzyloxycarbonyl: benzyloxycarbonyl; Cl-Z, 2-chlorobenzyloxycarbonyl: 2-chlorobenzyloxycarbonyl; Fm, 9-fluorenylmethyl: 9 - mercaptomethyl; Fmoc, 9-fluorenylmethoxycarbonyl: 9-fluorenylmethoxycarbonyl; Mtt, 4-methyltrityl: 4-methyltrity
  • ACN acetonitrile: acetonitrile
  • BOP benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate: benzotriazole-1-tris(trimethylamino)-hexafluorophosphate (Carter condensate);
  • DCC N, N'-Dicyclohexylcarbodiimide: Dicyclohexylcarbodiimide; DCM: dichloromethane;
  • DEPBT 3- (Diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one: 3-(diethoxyo-acyloxy)-1,2,3-benzotriazin-4-one;
  • DIC N , N'-Diisopropylcarbodiimide: N,N'-diisopropylcarbodiimide
  • DIPEA or DIEA
  • diisopropylethylamine diisopropy
  • PEG20K 4-arm and PEG40K 8-arm reagents were purchased from NOF America.
  • the reagents used in the present invention are all from commercial sources, or conventionally formulated reagents in the art, and the experiments and the steps thereof can be accomplished by those skilled in the art in light of the present invention and conventional techniques in the art.
  • Linear polypeptides use Boc solid phase peptide synthesis or Fmoc solid phase peptide synthesis. If Fmoc is used to synthesize a polypeptide having a C-terminal carboxyl group, Wang resin is used; and a polypeptide having a C-terminal amide is selected from Rink amide resin. If Boc chemistry is used to synthesize a polypeptide having a C-terminal carboxyl group, a Pam resin is used; and a polypeptide having a C-terminal amide is an MBHA resin.
  • the condensing agent and activator are DIC and HOBT, and other optional peptide bond condensing agents include EDC, BOP, HBTU, DEPBT and the like. A 5-fold excess of amino acids. The condensation time was 1 hour.
  • the Fmoc protecting group was removed with 50% piperidine/DMF.
  • the Boc protecting group was removed using TFA.
  • the peptide bond condensation reaction was monitored with a ninhydrin reagent.
  • the commonly used cleavage reagent is TFA.
  • the dry resin was placed in a shake flask, and an appropriate amount of trifluoroacetic acid/1,2-ethanedithiol/water (95:2.5:2.5, 10-25 mL/g resin) was added, and after shaking for 2 hours, the resin was filtered. The filtrate was added with 8-10 volumes of ice diethyl ether. Finally, the precipitated polypeptide was collected by centrifugation.
  • Boc-Cys(4-MeBzl)-OH Boc-Asp(OcHx)-OH, Boc-Glu(OcHx)-OH, Boc-His(Bom)-OH, Boc-Lys(2-Cl-Z)-OH , Boc-Asn(Xan)-OH, Boc-Arg(Tos)-OH, Boc-Ser(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Trp(CHO)-OH and Boc-Tyr( 2-Br-Z)-OH.
  • PAM and MBHA resins are generally cut with HF, 5 ml of HF per 0.1 mmol of resin, additional p-cresol, p-nonylphenol or anisole, and the mixture is stirred for 1 hour in an ice bath. . After HF vacuum extraction, the polypeptide was precipitated with ice diethyl ether and the precipitated polypeptide was collected by centrifugation.
  • Boc-Lys(Fmoc)-OH (187 mg, 400 ⁇ mole), DEPBT (120 mg, 400 ⁇ mole), DIPEA (140 ⁇ L, 800 ⁇ mole) was added to a solution of PEG20K-(NH 2 ) 4 (2 g, 100 ⁇ mole) in dichloromethane (30 mL). Stir at room temperature for 4 hours. Acetic anhydride (50 ⁇ L, 526 ⁇ mole) was added to the reaction, and stirring was continued for 15 minutes. The reaction solution was concentrated to 10 mL, added with MTBE (100 mL) and stirred for 15 min. The two batches of the reaction products were combined and used directly in the next reaction.
  • DBCO-OSu (81.2 mg, 200 ⁇ mole) and DIPEA (69 ⁇ L, 400 ⁇ mole) were added [Boc-Lys-NH] 4 -PEG20K (837 mg, 40 ⁇ mole) in acetonitrile (8 mL). The reaction was stirred at room temperature for 2 hours, then acetic anhydride (20 ⁇ L, 210.4 ⁇ mole) was added and stirring was continued for 20 min. After removing the solvent under reduced pressure, the solid was purified by HPLC to give [Boc-Lys(DBCO)-NH] 4 - PEG 20K (776 mg).
  • PEG40K(NH 2 ) 8 (2 g, 50 ⁇ mole) was dissolved in acetonitrile (50 mL), EtOAc (EtOAc) (EtOAc) The acetonitrile was evaporated to 10 mL under reduced pressure. The products of the two batches were combined, added to an aqueous solution containing 0.1% TFA, and purified by preparative HPLC. A solution: 0.1% TFA B solution: 0.1% TFA, 100% acetonitrile to give PEG 40K (NH-Fmoc) 4 (NH 2 ) 4 (0.83g). Other incorrect products can be removed from the Fmoc and recycled for reuse.
  • PEG40K(NH-Fmoc) 4 (NH 2 ) 4 (0.8 g) was dissolved in acetonitrile (5 mL).
  • EtOAc (36.2 mg) and DIEA (35 ⁇ L) were added and stirred at room temperature for 3 hours.
  • the reaction was purified by dialysis (Slide-A-lyzer Dialysis Cassette, 20K MWCO, Thermo Fisher Scientific) to give PEG40K(NH-Fmoc) 4 (DBCO) 4 (0.71 g).
  • PEG40K(NH-Fmoc) 4 (DBCO) 4 (0.71 g) was dissolved in DMF (6 mL). Of MTBE was added dropwise to the reaction, the precipitate was filtered, washed with 3 times of MTBE, and dried in vacuo to give a white solid PEG40K (NH 2) 4 (DBCO ) 4 (0.6g).
  • the hydrogel (50 mg) obtained in Example 5 was suspended in acetonitrile (200 ⁇ L), and DBCO-OSu (4 mg, 10 ⁇ mol) and DIEA (2 ⁇ L, 11.5 ⁇ mol) were added. After 12 hours, the supernatant was removed and the hydrogel was washed with acetonitrile (2 ⁇ 1 mL). Resuspended in acetonitrile (200 ⁇ L), DIEA (4 ⁇ L, 23 ⁇ mol) and acetic anhydride (2.2 ⁇ L, 23 ⁇ mol) were added, and after 1 hour, the supernatant was removed and dried. 50% TFA/CH 2 Cl 2 (200 ⁇ L) was added to the hydrogel and removed after 15 minutes.
  • DBCO connection between such substituted and backbone hydrogel crosslinked as in Figure 3B may be N 3 - biodegradable linker - biologically active substances reaction to be administered hydrogel.
  • the hydrogel (50 mg) obtained in Example 5 was suspended in DMF (500 ⁇ L), and MAL-dPEG 4 -NHS (Quanta Biodesign, 5.3 mg, 20 ⁇ mol) and DIEA (4 ⁇ L, 23 ⁇ mol) were added. After 30 minutes, the supernatant was removed, washed successively with purified water (1 mL), ethanol (1 mL) and acetonitrile (1 mL), and dried in vacuo to give a hydrogel.
  • the structure of the mixture between the skeleton and the crosslinked product was as shown in Fig. 3C. . .
  • the water-hydrogel suspension was wet-sifted on 200, 300, 360 and 500 mesh steel screens, separately collected, washed successively with water, ethanol and acetonitrile, then suspended in DMF (5 mL), and piperidine (1 mL) was added. After 15 minutes, the supernatant was decanted, washed with dichloromethane, water and ethanol, and dried under vacuum to obtain a white powdery hydrogel bead.
  • the structure of the skeleton and the crosslinked product was as 4D, in which the free amino group could continue to be derivatized. Other orthogonal reaction functional groups.
  • This hydrogel contains two different reactive functional groups that can be used for different purposes, such as loading two different biologically active substances.
  • the Boc protecting group on the degradable linking group can be cleaved with TFA during subsequent synthesis. Increasing the reactive functional group in the cross-linking can increase the drug loading compared to 3C.
  • N-bromosuccinimide 38.9 g, 0.22 mol
  • benzoyl peroxide 1.1 g, 4.4 mmol
  • 6D 50 g, 0.22 mol
  • CCl 4 solution 750 mL
  • the reaction mixture was stirred at 60 ° C for 4 hours under a 250 W lamp.
  • the mixture was cooled to room temperature, washed with aq. Evaporation of the solvent gave crude 6E (75 g) as an oil which was used directly in the next step.
  • Human insulin (18 mg, 3.1 ⁇ mol) and 7E (2 mg, 2.57 ⁇ mol) were dissolved in DMSO (300 ⁇ L), DIEA (1.56 ⁇ L, 9 ⁇ mol) was added and stirred for 1 hour. Purified by RP-HPLC. The lyophilized solid was added to TFA (2 mL), stirred for 15 minutes, added to a 20% acetonitrile solution (5 mL), purified by RP-HPLC and lyophilized.
  • exenatide is synthesized by the above-mentioned polypeptide synthesis method, and purified by RP-HPLC.
  • This Exenatide prodrug can be reacted with a hydrogel containing a DBCO reactive group and loaded onto a hydrogel.
  • Polypeptide GIVEQAA-NH 2 and GIVEQAAY two analog insulin A chain N-terminal portion of the polypeptide used as a model to measure the amide linkage may be a variety of N-terminal degradation half polypeptide linking group.
  • the peptide model was synthesized by Fmoc chemistry, cleaved with TFA, and purified by preparative HPC.
  • the polypeptide model blocked by the degradable linking group was dissolved in physiological saline at a concentration of 1 mg/mL, and cultured in a 37 ° C water bath. Samples were sampled at different time points (eg, 8 h, 16 h, 24 h, 48 h, 72 h, 96 h, 120 h). If the half-life of a polypeptide model terminated by a degradable linking group is significantly prolonged or shortened, the time frame for sampling analysis will change accordingly. The addition of 0.1% TFA solution reduced the pH to 2 to inhibit the degradable linking group from breaking. RP-HPLC was used to detect the cleavage reaction. The rate of fragmentation was quantified by measuring the area of the peaks of the polypeptide model capped and released by the degradable linker. Agilent 6110 quadrupole LC-MS was used to confirm mass changes during the reaction.
  • RP-HPLC analysis was performed using a Shimadzu LC-2010A HT system and a 150 mm x 4.6 mm Zorbax 300SB-C18 column. The flow rate was 1 ml/min. Solvent A contained 0.1% TFA / 5% CH 3 CN deionized water and solvent B contained 0.1% TFA 100% CH 3 CN. A linear gradient (eg 10-70% B, 10 minutes) is used.
  • the rate of cleavage of each of the degradable linker-terminated polypeptide models was determined.
  • the concentration of the degradable linker-terminated polypeptide model and the released polypeptide model were determined using their respective peak areas.
  • the first-order separation rate constant of a degradable linker-terminated peptide model by plotting the logarithm of the [starting degradable linker-terminated peptide model]/[residual degradable linker-terminated peptide model] at different time points To determine.
  • the rupture half-life of the degradable linker-terminated peptide model is calculated by using the formula and slope obtained from the plot.
  • the cleavage half-life of the various degradable linking groups attached to the polypeptide model can be determined using the procedures described above. The data generated by these tests are shown in Tables 1 and 2.
  • the cleavage half-life of the following compounds was 779 hours.
  • a peptide model X-G-dI-dV-dE-dQ-dA-dA terminated with a degradable linking group was used in the analysis.
  • Type D amino acids are used to avoid digestion of the polypeptide model.
  • the polypeptide was dissolved in 100% serum and incubated at 37 °C. Samples were sampled at different time points (eg, 8 h, 16 h, 24 h, 48 h, 72 h, 96 h, 120 h). If the half-life of a polypeptide model terminated by a degradable linking group is significantly prolonged or shortened, the time frame for sampling analysis will change accordingly.
  • the rate of cleavage of each of the degradable linker-terminated polypeptide models was determined.
  • the concentration of the degradable linker-terminated polypeptide model and the released polypeptide model were determined using their respective peak areas.
  • the first-order separation rate constant of a degradable linker-terminated peptide model by plotting the logarithm of the [starting degradable linker-terminated peptide model]/[residual degradable linker-terminated peptide model] at different time points To determine.
  • the rupture half-life of the degradable linker-terminated peptide model is calculated by using the formula and slope obtained from the plot.
  • the hydrogel (5 mg) obtained in Example 7 was suspended in PBS, and 8A (6 mg) was added thereto. After reacting for 30 minutes at room temperature, the supernatant was removed and washed with PBS to obtain a hydrogel containing insulin, and dried under vacuum. 50% TFA/CH 2 Cl 2 (200 ⁇ L) was added to the hydrogel and removed after 15 minutes. The hydrogel was washed with acetonitrile (3 x 1 mL) and purified water.
  • an insulin hydrogel (1 mg) was taken and the insulin on the hydrogel was completely released from the hydrogel in a water bath at pH 10.
  • the insulin content in the sample was calculated by comparing the area of the insulin peak in HPLC with the area of the known precisely quantified insulin standard under the same system, with an actual insulin content of 0.3 mg.
  • the hydrogel containing insulin (1 mg) was suspended in PBS (1 ml) in a water bath at 37 °C. Sample every 48 hours.
  • the release half-life of insulin in physiological saline was measured according to the method in Example 13. The release half-life of insulin is 197 hours.
  • Example 6 The hydrogel (10 mg) obtained in Example 6 was suspended in PBS (2 ml), and 9A (5 mg) was added. After 1 hour, the supernatant was removed and washed with purified water to obtain a hydrogel loaded with exenatide.
  • the exenatide content, exenatide release half-life measurement and calculation method of the exenatide hydrogel were the same as those in Example 18. Each milligram of hydrogel contains 0.2 milligrams of exenatide. Exenatide has a half-life of 143 hours.
  • the hydrogel (100 mg) obtained in Example 8 was suspended in dichloromethane (500 ⁇ L), and MAL-dPEG 4 -NHS (Quanta Biodesign, 1.59 mg) and DIEA (2 ⁇ L) were added. Thirty minutes later, TFA (500 ⁇ L) was added, and after 20 minutes, the supernatant was removed, and the hydrogel was washed with dichloromethane (2 ⁇ 1 mL), acetonitrile (2 ⁇ 1 mL) and physiological saline (2 ⁇ 1 mL), and then suspended in physiological saline (500 ⁇ L).
  • Recombinant urate oxidase lyophilized powder (1 mg), 10B (20 mg) and PEG5K-[NHC(O)-CH 2 CH 2 N 3 ] 8 (10 mg) were dissolved in physiological saline (400 ⁇ L), and allowed to stand for 5 hours.
  • HPLC analysis showed no free urate oxidase in the supernatant indicating that the urate oxidase had been encapsulated by the hydrogel.
  • Human serum 600 ⁇ L was added to the urate oxidase hydrogel, and cultured in a 37 ° C water bath, and timed by HPLC, using a Phenomenex Bio-Sep SEC-S2000 4.6X 300 mm 5 ⁇ M gel filtration column.
  • the time for complete release of urate oxidase was 416 hours.
  • Labrase (recombinant urate oxidase) is a short-acting drug administered by intravenous drip.
  • the urate oxidase hydrogel can be formulated as a long-acting dosage form for subcutaneous injection by the patient himself.
  • the molecular weight (the 8-arm PEG in this example) is adjusted by the molecular weight of the cross-linking material, the cross-linking density, and the like.
  • the larger the molecular weight of the skeleton the longer the length of the crosslinked product, the larger the pore size of the hydrogel, and the encapsulated protein is more easily permeated.
  • Crosslink density is also an effective method of adjusting the pore size of the hydrogel.
  • Fmoc-Lys(Mtt)-OH 250 mg, 400 ⁇ mole
  • DEPBT 120 mg, 400 ⁇ mole
  • DIPEA 140 ⁇ L, 800 ⁇ mole
  • PEG20K-(NH 2 ) 4 2 g, 100 ⁇ mole
  • dichloromethane 25 mL
  • acetic anhydride 50 ⁇ L, 526 ⁇ mole
  • the reaction solution was concentrated to 10 mL, added with MTBE (80 mL) and stirred for 10 min.
  • the solid precipitate was filtered and dried in vacuo and applied directly to the next step.
  • the hydrogel 23A (20 mg) obtained in Example 23 was suspended in physiological saline, and 8A (20 mg) was added thereto, and the reaction was allowed to stand overnight. The supernatant was removed and washed with physiological saline (2 ⁇ 3 mL) to obtain a hydrogel 23A-insulin containing insulin.
  • the hydrogel 23B (20 mg) was suspended in physiological saline, 8A (20 mg) was added, and the reaction was allowed to stand overnight, and the supernatant was removed, and washed with physiological saline (2 ⁇ 3 mL) to obtain a hydrogel 23B-insulin containing insulin.
  • Hyaluronic acid (MW: 100K, 200 mg) was dissolved in 0.1 M MES buffer (100 mL), EDC (144 mg, 0.75 mmole) and N-hydroxysuccinimide (86 mg, 0.75 mmole) were added and stirred for 30 minutes.
  • N-(2-Aminoethyl)maleimide hydrochloride (110 mg, 0.63 mmole) was dissolved in 0.1 M MES buffer (40 mL) and then reacted with hyaluronic acid.
  • the 1 H-NMR (D 2 O) spectrum of hyaluronic acid-maleimide showed a peak of ⁇ 7.0, corresponding to the vinyl proton of maleimide.
  • the degree of substitution of hyaluronic acid-maleimide is defined as the amount of maleimide per 100 hyaluronic acid disaccharide rings, by comparison to hyaluronic acid acetamide methyl protons and maleimide The vinyl protons were determined by ⁇ 2.0 and ⁇ 7.0.
  • the degree of substitution measured by the above reaction was 8. By reducing the ratio of N-(2-aminoethyl)maleimide to hyaluronic acid in the reaction, the degree of substitution can be lowered.
  • Hyaluronic acid-maleimide and PEG10K-(SH) 4 were dissolved in physiological saline, 1-3% w/v, and the ratio of thiol to maleimide was 1.1. As the concentration increases, the gel time is shortened. When the degree of substitution is 4, the gel time of 1% w/v is 40 s, and the gel time of 3% w/v is 15 s. As the degree of substitution increases, the gel time also decreases. When the degree of substitution is 8, the 3% w/v gel time is 7 s.
  • Hyaluronic acid-maleimide, PEG10K-(SH) 4 and 8A are dissolved in physiological saline, maleimide: PEG thiol: 8A thiol ratio 1:0.8:0.3, 1% w/v, mix After standing for 1 hour, hyaluronic acid-maleimide-8A was obtained, which can be administered by subcutaneous injection.
  • mice of 9 weeks old, with an average body weight of 240 ⁇ 10 g were placed in a polypropylene cage, kept at a constant temperature and humidity, 12 hours light/dark cycle, and freely ingested food and water.
  • the rats were fasted for 48 hours, intraperitoneally injected with streptozotocin (60 mg/kg, dissolved in 10 nM sodium citrate buffer, pH 4.5), and then food was provided.
  • streptozotocin 60 mg/kg, dissolved in 10 nM sodium citrate buffer, pH 4.5
  • Food was provided.
  • Rats with blood glucose above 250 mg/dl were defined as diabetes, and rats with blood glucose above 450 mg/dl were used for the experiment.
  • Recombinant insulin 24 nanomoles/kg was injected daily to maintain blood glucose levels not exceeding 550 mg/dl.
  • the insulin hydrogel was prepared as in Example 8 and Example 20, but the hydrogel used in this example was loaded with only insulin.
  • the actual insulin content was measured by the method of Example 18.
  • Rats with hyperglycemia were divided into 3 groups of 8 animals each.
  • the first group of rats was intraperitoneally injected with normal saline (100 ⁇ L), and the second group of rats was intraperitoneally injected with 40 nmol/kg of human insulin per day.
  • the third group of rats was injected with hydrogel (75 nanomoles/kg, calculated according to the actual amount of human insulin in the hydrogel).
  • Another group of 8 healthy rats were intraperitoneally injected with normal saline (100 ⁇ L) as a control. Measured at 11 am. The result is shown in Figure 1.
  • Detemir is a representative long-acting insulin, which is used once a day for clinical use. Insulin hydrogel is injected subcutaneously once, and the hypoglycemic effect is maintained for at least one week. Detemir is a long-acting modification of fatty acids and has a reduced biological activity, so it is not effective in lowering blood glucose at a dose of 40 nanomoles/kg. The insulin released by the insulin hydrogel retains all of its activity, lowering blood glucose to a normal value at a lower dose than detemir. In addition, insulin hydrogel does not exhibit the "burst" phenomenon at the beginning as many other hydrogels, and there is no hypoglycemia caused by excessive insulin. The blood glucose of the insulin hydrogel group was almost steady in 1-7 days, indicating that insulin was released at a uniform rate. This example illustrates that the hydrogel of the present invention can be an effective long-acting drug delivery technique.

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Abstract

一种可在生理条件下降解的水凝胶，所述水凝胶包括至少一种骨架和交联物，所述交联物中包括可降解连接基团，所述可降解连接基团通过分子内环化反应降解。

Description

一种可在生理条件下降解的水凝胶 技术领域
本发明涉及生物工程领域，具体而言，涉及一种可在生理条件下降解的水凝胶。
背景技术
水凝胶是有潜力的给药系统材料。水凝胶的高含水量使水凝胶具有生物相容性，减少与其接触的组织的炎性反应。水凝胶适用于小分子和大分子药物，水凝胶提供充分含水的环境，尤其适合保持多肽、蛋白、低(聚)核苷酸或者多(聚)核苷酸药物的生物活性和结构完整。
装载生物活性物质的水凝胶在给药后，可以在患者体内形成储存库(depot)，在期待的时间长度内持久释放生物活性物质。根据生物活性物质与水凝胶的连接方式，有两种制备基于水凝胶的储存库的方法：非共价的和共价的储存库。
在非共价储存库方法中，生物活性物质例如药物通过物理方法而不是通过化学键封装在水凝胶内。在这种方法中，水凝胶的三维网络中的平均孔径必须小于生物活性物质的大小，从而保证水凝胶有效封装。因此，生物活性物质不能在水凝胶形成后再被加入。在非共价方法中，水凝胶必须在生物活性物质存在的情况下化学交联，或者在生物活性物质存在的情况下，通过自组装过程的物理交联形成孔。水凝胶可以通过交联亲水性生物聚合物或合成聚合物制备。亲水性生物聚合物通过物理或化学交联形成水凝胶的例子包括但不限于透明质酸、壳聚糖、藻酸盐、胶原、葡萄聚糖、果胶、卡拉胶、多聚赖氨酸、明胶或琼脂糖等。由合成聚合物的化学或者物理交联形成的水凝胶的例子包括聚乙二醇、聚丙二醇、聚(乳酸-乙二醇共聚物)酸(PLGA)聚合物等。在非共价储存库方法中，水凝胶的三维网络中的孔径随着水凝胶逐步降解而扩大，封装的生物活性物质因此游离出来。
在共价储存库方法中，生物活性物质通过可逆或可降解连接基团连接到水凝胶上。
无论是非共价储存库方法还是共价储存库方法，现有绝大多数水凝胶的降解依赖酶催化。酶水平和专一性在病人群中，甚至同一病人不同健康状态下差异很大，而且依赖于选作注射点的组织和其它难以控制的参数。这种实际给药与预期之间的差异影响治疗效果，治疗指数低的药物的毒副作用增加。
另外一个造成复杂性的事实在于聚合物在体内条件下降解也可以是化学降解。通常用作可生物降解键的酯键可能在生理pH 7.4，没有酶的纯缓冲液中自发水解。为了有效释放生物活性物质，很多水凝胶包含大量酯键。局部高密度的酯键和紧密封装的生物活性物质都可以导致副反应。生物活性物质中的氨基有可能处于靠近酯键的位置，氨基提供亲核试剂，促进酯键断开和随后的酰胺化。这个过程导致生物活性物质与聚合物之间形成非常稳定的酰胺键连接。生物活性物质在它连接的聚合物链被降解之前不会被释放出来，而且生物活性物质被永久修改了。已知这种修改降低生物活性物质的生物活性，可能会导致副作用。此外，这种不可取的修改过程基本不可控制，造成多种分子结构。
给药完成后，水凝胶需要从体内清除。通过手术去除水凝胶往往给患者增加痛苦。尽管有些已知的水凝胶声称可以生物降解，但所述降解不受控制并因此不可预知。
发明内容
本发明在水凝胶骨架内部，或者水凝胶骨架之间的交联物中引入可降解连接基团。这些可降解连接基团能够在生理条件下(例如，37℃，pH值7.0-7.6)通过化学反应自动断裂，速度取决于温度和pH，不需要酶或者其它试剂辅助。由于生理条件在人群中通常恒定，这种新型水凝胶能以预定速率生物降解，给药在病人个体内和个体之间的差异很小，产生更一致的治疗效果。另外，这种新型水凝胶能够完全降解成为小分子量片段，从体内清除，不会在注射部位或者体内积聚。
本发明提供在生理条件下以非酶过程降解成为水溶性的、较小分子量成份的水凝胶，以及所述水凝胶的制备方法。水凝胶的骨架或者交联物包含可降解连接基团，在生物条件下断裂，从而导致水凝胶降解。本发明还涉及可降解连接基团本身，以及本发明形成过程中的中间体。
本发明的可降解连接基团包括可以在生理条件下自动断裂的部分，还包括与反应性聚合物形成共价键的反应基团。本发明中的反应性聚合物可以是水凝胶骨架或者交联物。所述可以在生理条件下自动断裂的部分具有式
Figure PCTCN2016110876-appb-000001
X、Y、Y1、A、B、J1在下文有详细定义。Y、A、B中至少包含一个可以与反应性聚合物形成共价键的反应基团。波浪线是可降解连接基团与生物活性物质或者反应性聚 合物形成酰胺键或者酯键的连接点。可降解连接基团将两种或多种反应性聚合物连接在一起。
根据本发明制备的水凝胶可以应用于给药系统、生物医学工程等不同领域。
根据本发明制备的水凝胶还可以包含一种或多种药物或者生物活性物质。所述一种或多种药物或者生物活性物质可以通过物理方法而不是通过化学键封装在水凝胶内部。药物或者生物活性物质也可以与可降解连接基团的一端连接，可降解连接基团的另一端与所述水凝胶偶联。可降解连接基团断裂后，药物或者生物活性物质可以独立释放，而不取决于水凝胶的降解。本发明还提供生物可降解给药水凝胶的制备方法，其中药物释放速率和水凝胶降解速率是可控的。
附图说明
图1显示了给糖尿病大鼠注射生理盐水、地特胰岛素和胰岛素水凝胶后大鼠在一周内的血糖变化。
具体实施方式
本发明的水凝胶是由在生理条件下可降解连接基团交联而成的聚合物。在生理pH和温度，水凝胶由于这些连接基团断裂而失去结构完整性，产生的碎片足够小而且可溶，通过常规的生物学通路从体内清除。
除非另有说明，下列术语的定义适用于整个专利。本文没有定义的术语具有该术语在相关文献的普遍理解的含义。
本发明中的“生物活性物质”是指治疗性或药理性物质。生物活性物质可以是小分子、多肽、蛋白、DNA、RNA、细胞等。生物活性物质的例子包括但不限于以下治疗领域的物质：ACE抑制剂；抗心绞痛药；抗心律失常药；抗哮喘药；抗胆甾醇药；抗惊厥剂；抗抑郁药；抗腹泻制剂；抗组胺剂；降压药；抗感染药物；消炎药；抗脂质剂；抗躁狂药；止恶心药；防中风药；抗甲状腺制剂；抗肿瘤药；止咳药；抗尿酸血症药；抗病毒剂；痤疮药；生物碱；氨基酸制剂；合成代谢药物；镇痛药；麻醉剂；血管生成抑制剂；抗酸剂；抗关节炎药；抗生素；抗凝血剂；止吐药；减肥药；抗寄生虫药；抗精神病药；退烧药；解痉药；抗血栓药；抗焦虑药；食欲刺激剂；抑制食欲剂；β受体阻断药；支气管扩张剂；心血管药；脑扩张剂；螯合剂；胆囊收缩素拮抗剂；化疗药物；认知激活剂；避孕药；冠状血管扩张剂；减充血剂；皮肤病药；糖 尿病药；利尿剂；润肤剂；酶；促红细胞生成药；祛痰剂；生育剂；杀菌剂；胃肠道药；生长调节剂；激素替代剂；血糖剂；催眠药；降糖药；泻药；偏头痛药；黏液溶解剂；抗精神病药；神经肌肉药物；非甾体抗炎药；周围血管扩张药；前列腺素；精神药物；肾素抑制剂；呼吸兴奋剂；类固醇；兴奋剂；交感神经药；甲状腺制剂；镇静剂；子宫松弛剂；阴道制剂；血管收缩剂；血管扩张剂；眩晕剂；维生素和伤口愈合剂。
本发明中，术语“可(生物)降解”或“自动断裂”是指在生理条件下不需要酶催化即可降解。这样的键包括但不局限于缩醛键、酯键、亚胺键、腙键、羧酸酐、乌头键、原酸酯键、马来酰胺酸酰胺键、磷酰胺键、磷酰酯键、磺酸酯键、芳族氨基甲酸酯键以及它们的组合等。优选的是羧酸酯键和碳酸酯键。但这些键的共同缺点是稳定性差，既容易被各种酶降解，也易于水解。以这些键为主的药用水凝胶的药物释放速率和水凝胶降解速率无法准确控制。
酰胺键以其稳定性出名，水解通常需要强酸(例如硫酸、盐酸)或强碱(例如氢氧化钠)和高温(例如100℃)。由于酰胺在体内的相对稳定性，不需要酶水解而可以自动断裂的酰胺结构非常罕见。本发明显示，利用分子内化学催化的优势，酰胺键可以在温和的条件下水解。设计和修改适当的分子骨架，以及选择这个骨架不同位置的空间、吸电子或者供电子取代基能够调节酰胺键在生理条件下的反应速率。同样的原理也适用于酯键。
本发明的可降解连接基团在生理条件下断裂的部分可以用以下通式表示：
Figure PCTCN2016110876-appb-000002
其中X选自-OH或-HN-R0；
Y选自：
(1)N-RO；
(2)C(RpRq)；
(3)O，条件是X不是OH；
(4)C(RpRq)，当X是-HN-R0，其中R0和Rp连同与其相连的原子可以组成4，5或6元杂环；
Y1选自
(1)C(R3R4)；
(2)C(O)或C(S)，条件是A不是C(O)，C(S)，SO或SO2；
(3)O，S，SO或SO2，条件是Y和A不是O；
(4)N-Rn；和
(5)键；
J1是-C(R10R11)或共价键。
A和B共同构成环系统，包括：有6-15个碳原子的芳基；有4，5，6，7，8，9或10元环的环烷基；有4，5，6，7，8，9，或10元环的环烯基；有5，6，7，8，9或10元环的环炔基；饱和和不饱和单环、多环和稠环；饱和和不饱和单杂环、多杂环和稠杂环，每个杂环包含一个或多个杂环原子N，S或O，而且每个环是4元环到10元环。前述的每一个环可选地被一个或多个基团取代，基团选自低级烷基(lower alkyl)、低级烷氧基(lower alkoxy)、酰基、酰氧基、烷氧羰基，芳基、苯基、苄基、卤素、卤代甲酰基、卤代低级烷基(haloloweralkyl)、三氟甲基、三氟甲氧基、三氯甲基、氰酰、异氰、异氰酸酯(或盐)、异硫氰酸酯、硫氰酸酯(或盐)、低级烷硫基(lower alkylthio)，氨基、亚氨基、氨基低级烷基(amino lower alkyl)、低级烷氨基(lower alkylamino)、低级烷氨基(lower dialkylamino)、羟基、羟烷基、硝基、腈、异腈、吡啶基、叠氮、羧基、羧酰氨基(carboxamido)、乙酸基、硫烷基(thiolalkyl)、碳酸酯(或盐)、氨基甲酸酯、低级烷氨基甲酰(loweralkylcarbamyl)、双低级烷氨基甲酰(diloweralkylcarbamyl)、磺酸基、磺胺、磺酸酯(或盐)、磺酰、亚砜、硫化物、二硫化物和巯基。
或者A、B及与其相连接的原子共同形成一个芳香环或芳杂环，可选地被至少一个以上定义的基团取代；或者A、B及与其相连接的原子共同组成一个多芳环或多芳杂环，可选地被至少一个以上定义的基团取代。
各种环的例子包括但不局限于，吖啶、氮杂环庚烷、吖庚因、杂环辛烷(azocane)、苯并呋喃、苯并咪唑、苯并噻吩、苯并[c]噻吩、苯并恶唑、苯异恶唑、苯并噻唑、邻二氮(杂)萘(cinnoline)、二氮杂草、二氧戊环、二硫戊环、呋喃、呋咱、咪唑烷、咪唑、吲唑、吲哚、异吲哚、异苯并呋喃、异噻唑、异噻唑烷、异恶唑、异恶唑烷、吗啉、恶嗪、恶二唑、恶唑、恶唑烷、氧杂环庚(oxepane)、喹唑啉、喹啉、异喹啉、喹喔啉、磷杂环戊二烯(phosphole)、酞嗪、哌啶、嘌呤、吡啶、吡喃、吡咯烷、吡咯、吡唑烷、吡嗪、吡唑、哌嗪、嘧啶、哒嗪、噻唑、四唑、1，2，3-三嗪、1，2，4-三嗪、1，3，5-三嗪、三唑、thiane、噻唑烷、噻喃、四氢呋喃、四氢噻吩、噻重氮、噻吩、硫代吗啉、噻嗪、硫氮杂(thiazepine)和zocine。
Y1(或者Y，如果Y1是共价键)和J1(或者C(O)，如果J1是共价键)分别与构成同一个环、被一个键分隔开的两个原子相连。
R0，RO，R3，R4，R10，R11，Rn，Rp，Rq各独立选自氢原子、低级烷基、低级烷氧基、酰基、酰氧基、烷氧羰基、芳基、苯基、苄基、卤素、卤代甲酰基、卤代低级烷基、三氟甲基、三氟甲氧基、三氯甲基、氰酰、异氰、异氰酸酯(或盐)、异硫氰酸酯、硫氰酸酯(或盐)、低级烷硫基、氨基、亚氨基、氨基低级烷基、低级烷氨基、低级烷氨基、羟基、羟烷基、硝基、腈、异腈、吡啶基、叠氮、羧基、羧酰氨基、乙酸基、硫烷基、 碳酸酯(或盐)、氨基甲酸酯、低级烷氨基甲酰、双低级烷氨基甲酰、磺酸基、磺胺、磺酸酯(或盐)、磺酰、亚砜、硫化物、二硫化物和巯基。或者R0，RO，R3，R4，R10，R11，Rn，Rp，Rq各独立选自-SO2-OH，-SO2-NRm1Rm2，-SO2-Rm3，-O-Rm4，-S-Rm5，-N-Rm6Rm7，-C(O)Rm8，-C(O)ORm9，-OC(O)Rm10，-NHC(O)Rm11，-C(O)NRm12Rm13，-NHC(O)NRm14Rm15，其中Rm1，Rm2，Rm3，Rm4，Rm5，Rm6，Rm7，Rm8，Rm9，Rm10，Rm11，Rm12，Rm13，Rm14和Rm15独立选自氢原子(H)、(C1-C18)烷基、芳基、(C1-C18烷基)OH、(C1-C18烷基)SH、(C1-C18烷基)COOH、(C1-C18烷基)NH2、(C0-C4烷基)(C5-C6环烷基)、(C0-C10烷基)(C5-C6杂环)、(C0-C4烷基)(C6-C10芳基)、(C0-C4烷基)(C4-C9杂芳基)。或者Rp和Rq各独立选自C1-C20烷基、(C1-C10烷基)OH、(C1-C10烷基)SH、(C2-C3烷基)SCH3、(C1-C4烷基)CONH2、(C1-C10烷基)COOH、(C1-C10烷基)NH2、(C1-C4烷基)NHC(NH2 +)NH2、(C0-C4烷基)(C3-C6环烷基)、(C0-C4烷基)(C2-C5杂环)、(C0-C4烷基)(C6-C10芳基)、(C0-C4烷基)(C6-C10芳基)R16、(C1-C4烷基)(C3-C9杂芳基)、C1-C12烷基(W1)C1-C12烷基，其中W1是选自N、S和O的一个杂原子，R16选自H、OH、卤素、C1-C7烷基、C2-C7烯基、C2-C7炔基、CO2H，CO2(C1-C7烷基)、NH(C0-C10烷基)、O(C1-C10烷基)、杂芳基。
本领域技术人员理解，含有一个或多个取代基的可降解连接基团，不引入在空间上不实际的、合成上不可行或内在不稳定的任何取代基或取代模式。
本发明中可降解连接基团的可降解部分通过分子内环化反应降解。
Figure PCTCN2016110876-appb-000003
其中R14和R15既可以是含氮或羟基的生物活性物质的一部分，也可以是含氮或羟基的反应性聚合物的一部分。
通过分子内环化反应降解不需要酶的参与，速率取决于温度和生理学pH。由于人群中体温和生理学pH基本一致，因此这种降解方式消除了酶降解中出现的人与人之间以及个人之间的差异，产生更加一致和可重复的治疗效果。
在某些实施方式中，J1是C(R10R11)，Y1是共价键，可降解连接基团的化学式如下：
Figure PCTCN2016110876-appb-000004
在某些实施方式中，Y1is C(R3R4)，J1是共价键，可降解连接基团的化学式如下：
Figure PCTCN2016110876-appb-000005
在某些实施方式中，Y1is C(R3R4)，J1是C(R10R11)，可降解连接基团的化学式如下：
Figure PCTCN2016110876-appb-000006
在某些实施方式中，可降解连接基团的化学式如下：
Figure PCTCN2016110876-appb-000007
子类1
在某些实施方式中，一类可降解连接基团的化学式如下：
Figure PCTCN2016110876-appb-000008
其中X选自-OH或-HN-R0；
Y选自：
(1)N-RO；
(2)C(RpRq)；
(3)O，条件是X不是OH；
(4)C(RpRq)，当X是-HN-R0，其中R0和Rp连同与其相连的原子可以组成4，5或6元杂环；
Y1选自
(1)C(R3R4)；
(2)C(O)或-C(S)，条件是Y和A不是C(O)，C(S)，SO或SO2；
(3)O，S，SO，or SO2，条件是Y和A不是O；
(4)N-Rn；和
(5)键；
J1是-C(R10R11)或共价键。
a、b和与其相连的原子共同构成双环系统，包括一个饱和5元环或6元环及与之稠合的芳环。饱和5元环或6元环可以是环烷基，也可以是杂环，杂环包含一个或多个杂环原子N，S或O。Y1(或者Y，如果Y1是共价键)和J1(或者C(O)，如果J1是共价键)分别与构成饱和5元环或6元环、被一个键分隔开的两个原子相连。芳环不与J， Y，C(O)，或Y直接相连。芳环的氢原子可选地被一个或多个基团取代，基团选自低级烷基、低级烷氧基、酰基、酰氧基、烷氧羰基，芳基、苯基、苄基、卤素、卤代甲酰基、卤代低级烷基、三氟甲基、三氟甲氧基、三氯甲基、氰酰、异氰、异氰酸酯(或盐)、异硫氰酸酯、硫氰酸酯(或盐)、低级烷硫基、氨基、亚氨基、氨基低级烷基、低级烷氨基、低级烷氨基、羟基、羟烷基、硝基、腈、异腈、吡啶基、叠氮、羧基、羧酰氨基、乙酸基、硫烷基、碳酸酯(或盐)、氨基甲酸酯、低级烷氨基甲酰、双低级烷氨基甲酰、磺酸基、磺胺、磺酸酯(或盐)、磺酰、亚砜、硫化物、二硫化物和巯基。
当饱和环是环烷基时，一个示范性的非限制性例子如下：
Figure PCTCN2016110876-appb-000009
其中m，n分别是0或1的整数，但m，n不同时为0。
在某些实施方式中，一类的可降解连接基团化学式如下，其中X，Y，R3，R4，R10，R11，a和b定义如上：
Figure PCTCN2016110876-appb-000010
a、b和与其相连的原子共同构成单芳环、多芳环、稠芳环、单杂芳环、多杂芳环或稠杂芳环，可选地被一个或多个基团取代，基团选自低级烷基、低级烷氧基、酰基、酰氧基、烷氧羰基，芳基、苯基、苄基、卤素、卤代甲酰基、卤代低级烷基、三氟甲基、三氟甲氧基、三氯甲基、氰酰、异氰、异氰酸酯(或盐)、异硫氰酸酯、硫氰酸酯(或盐)、低级烷硫基、氨基、亚氨基、氨基低级烷基、低级烷氨基、低级烷氨基、羟基、羟烷基、硝基、腈、异腈、吡啶基、叠氮、羧基、羧酰氨基、乙酸基、硫烷基、碳酸酯(或盐)、氨基甲酸酯、低级烷氨基甲酰、双低级烷氨基甲酰、磺酸基、磺胺、磺酸酯(或盐)、磺酰、亚砜、硫化物、二硫化物和巯基。
子类2
在某些实施方式中，一类包含6元环的可降解连接基团化学式如下：
Figure PCTCN2016110876-appb-000011
其中U1，U2，U3，U4，U5和U6独立选自CR12和N；
其中X选自-OH或-HN-R0；
Y选自：
(1)N-RO；
(2)C(RpRq)；
(3)O，条件是X不是OH；
(4)C(RpRq)，当X是HN-R0，其中R0和Rp连同与其相连的原子可以组成4，5或6元杂环；
Y1选自
(1)C(R3R4)；
(2)C(O)or-C(S)，条件是Y和A不是C(O)，C(S)，SO或SO2；
(3)O，S，SO或SO2，条件是Y和A不是O；
(4)N-Rn；和
(5)键；
J1是-C(R10R11)或共价键。
R12选自氢原子、低级烷基、低级烷氧基、酰基、酰氧基、烷氧羰基、芳基、苯基、苄基、卤素、卤代甲酰基、卤代低级烷基、三氟甲基、三氟甲氧基、三氯甲基、氰酰、异氰、异氰酸酯(或盐)、异硫氰酸酯、硫氰酸酯(或盐)、低级烷硫基、氨基、亚氨基、氨基低级烷基、低级烷氨基、低级烷氨基、羟基、羟烷基、硝基、腈、异腈、吡啶基、叠氮、羧基、羧酰氨基、乙酸基、硫烷基、碳酸酯(或盐)、氨基甲酸酯、低级烷氨基甲酰、双低级烷氨基甲酰、磺酸基、磺胺、磺酸酯(或盐)、磺酰、亚砜、硫化物、二硫化物和巯基。或者R12选自-SO2-OH，-SO2-NRm1Rm2，-SO2-Rm3，-O-Rm4，-S-Rm5，-N-Rm6Rm7，-C(O)Rm8，-C(O)ORm9，-OC(O)Rm10，-NHC(O)Rm11，-C(O)NRm12Rm13，-NHC(O)NRm14Rm15，其中Rm1，Rm2，Rm3，Rm4，Rm5，Rm6，Rm7，Rm8，Rm9，Rm10，Rm11，Rm12，Rm13，Rm14和Rm15独立选自氢原子(H)、(C1-C18)烷基、芳基、(C1-C18烷基)OH、(C1-C18烷基)SH、(C1-C18烷基)COOH、(C1-C18烷基)NH2、(C0-C4烷基)(C5-C6环烷基)、(C0-C10烷基)(C5-C6杂环)、(C0-C4烷基)(C6-C10芳基)、(C0-C4烷基)(C4-C9杂芳基)。
在某些实施方式中，R12包含羟基、氨基、羧基、巯基等功能团，可以与反应性聚合物的交联功能团形成共价键连接。在某些实施方式中，R12包含叠氮化物或炔基，可以通过点击化学与反应性聚合物上对应的炔基或叠氮化物反应。
子类3
在某些实施方式中，一类包含6元环的可降解连接基团化学式如下：
Figure PCTCN2016110876-appb-000012
其中X选自-OH或-HN-R0；
Y选自：
(1)N-RO；
(2)C(RpRq)；
(3)O，条件是X不是OH；
(4)C(RpRq)，当X是-HN-R0，其中R0和Rp连同与其相连的原子可以组成4，5或6元杂环；
U1，U2，U3，U4，U5，U6，R0，Ro，Rp和Rq的定义如上。
此类中的引入基团包括但不局限于以下例子：
Figure PCTCN2016110876-appb-000013
在某些实施方式中，以上3个化学式中的Y选自NH，N-RO，O(条件是X不是OH或SH)。
在某些实施方式中，Y是N-RO，X是HN-R0或OH。这类可降解连接基团化学式如下：
Figure PCTCN2016110876-appb-000014
在某些实施方式中，RO是氢原子(H)，这类可降解连接基团化学式如下：
Figure PCTCN2016110876-appb-000015
R5、R6、R7和R8独立选自氢原子、低级烷基、低级烷氧基、酰基、酰氧基、烷氧羰基、芳基、苯基、苄基、卤素、卤代甲酰基、卤代低级烷基、三氟甲基、三氟甲氧基、三氯甲基、氰酰、异氰、异氰酸酯(或盐)、异硫氰酸酯、硫氰酸酯(或盐)、低级烷硫基、氨基、亚氨基、氨基低级烷基、低级烷氨基、低级烷氨基、羟基、羟烷基、硝基、腈、异腈、吡啶基、叠氮、羧基、羧酰氨基、乙酸基、硫烷基、碳酸酯(或盐)、氨基甲酸酯、低级烷氨基甲酰、双低级烷氨基甲酰、磺酸基、磺胺、磺酸酯 (或盐)、磺酰、亚砜、硫化物、二硫化物和巯基。或者R5、R6、R7和R8独立选自-SO2-OH，-SO2-NRm1Rm2，-SO2-Rm3，-O-Rm4，-S-Rm5，-N-Rm6Rm7，-C(O)Rm8，-C(O)ORm9，-OC(O)Rm10，-NHC(O)Rm11，-C(O)NRm12Rm13，-NHC(O)NRm14Rm15，其中Rm1，Rm2，Rm3，Rm4，Rm5，Rm6，Rm7，Rm8，Rm9，Rm10，Rm11，Rm12，Rm13，Rm14和Rm15独立选自氢原子(H)、(C1-C18)烷基、芳基、(C1-C18烷基)OH、(C1-C18烷基)SH、(C1-C18烷基)COOH、(C1-C18烷基)NH2、(C0-C4烷基)(C5-C6环烷基)、(C0-C10烷基)(C5-C6杂环)、(C0-C4烷基)(C6-C10芳基)、(C0-C4烷基)(C4-C9杂芳基)。
在某些实施方式中，Y是O，这类可降解连接基团化学式如下，其中R5、R6、R7和R8定义如上：
Figure PCTCN2016110876-appb-000016
在某些实施方式中，Y是C(RpRq)。此类可降解连接基团化学式如下，其中X，U1，U2，U3，U4，U5和U6定义如上：
Figure PCTCN2016110876-appb-000017
在某些实施方式中，此类可降解连接基团包括，但不局限于以下例子，其中X、R5、R6、R7和R8定义如上：
Figure PCTCN2016110876-appb-000018
R9选自氢原子、低级烷基、低级烷氧基、酰基、酰氧基、烷氧羰基、芳基、苯基、苄基、卤素、卤代甲酰基、卤代低级烷基、三氟甲基、三氟甲氧基、三氯甲基、氰酰、异氰、异氰酸酯(或盐)、异硫氰酸酯、硫氰酸酯(或盐)、低级烷硫基、氨基、亚氨基、氨基低级烷基、低级烷氨基、低级烷氨基、羟基、羟烷基、硝基、腈、异腈、吡啶基、叠氮、羧基、羧酰氨基、乙酸基、硫烷基、碳酸酯(或盐)、氨基甲酸酯、低级 烷氨基甲酰、双低级烷氨基甲酰、磺酸基、磺胺、磺酸酯(或盐)、磺酰、亚砜、硫化物、二硫化物和巯基。或者R9选自-SO2-OH，-SO2-NRm1Rm2，-SO2-Rm3，-O-Rm4，-S-Rm5，-N-Rm6Rm7，-C(O)Rm8，-C(O)ORm9，-OC(O)Rm10，-NHC(O)Rm11，-C(O)NRm12Rm13，-NHC(O)NRm14Rm15，其中Rm1，Rm2，Rm3，Rm4，Rm5，Rm6，Rm7，Rm8，Rm9，Rm10，Rm11，Rm12，Rm13，Rm14和Rm15独立选自氢原子(H)、(C1-C18)烷基、芳基、(C1-C18烷基)OH、(C1-C18烷基)SH、(C1-C18烷基)COOH、(C1-C18烷基)NH2、(C0-C4烷基)(C5-C6环烷基)、(C0-C10烷基)(C5-C6杂环)、(C0-C4烷基)(C6-C10芳基)、(C0-C4烷基)(C4-C9杂芳基)。
在某些实施方式中，R5、R6、R7、R8和R9包含羟基、氨基、羧基、巯基等功能团，可以与反应性聚合物的交联功能团形成共价键连接。在某些实施方式中，R5、R6、R7、R8和R9包含叠氮化物或炔基，可以通过点击化学与反应性聚合物上对应的炔基或叠氮化物反应。
在某些实施方式中，Rp和Rq是氢原子(H)。此类中的一组可降解连接基团化学式如下。其中X，R5，R6，R7和R8定义如上：
Figure PCTCN2016110876-appb-000019
在某些实施方式中，这组的可降解连接基团的化学式如下：
Figure PCTCN2016110876-appb-000020
在某些实施方式中，此类可降解连接基团化学式如下，其中X，R6和R7定义如上：
Figure PCTCN2016110876-appb-000021
在某些实施方式中，本组引入基团化学式如下，其中X，R6和R7定义如上：
Figure PCTCN2016110876-appb-000022
在某些实施方式中，X在以上化学式中是OH或HN-R0。
在某些实施方式中，X在以上化学式中是OH。
在某些实施方式中，X在以上化学式中是HN-R0。
在某些实施方式中，X在以上化学式中是NH2。
本组可降解连接基团的图解的、非限制性例子包括：
Figure PCTCN2016110876-appb-000023
Figure PCTCN2016110876-appb-000024
子类4
在某些实施方式中，Y1是C(R3R4)。一类包含6元环的可降解连接基团化学式如下：
Figure PCTCN2016110876-appb-000025
其中X选自-OH或-HN-R0；
Y选自：
(1)N-RO；
(2)C(RpRq)；
(3)O，条件是X不是OH；
(4)C(RpRq)，当X是-HN-R0，其中R0和Rp连同与其相连的原子可以组成4，5或6元杂环；
U1，U2，U3，U4，U5，U6，R3，R4，R0，Ro，Rp和Rq的定义如上。
在某些实施方式中，此类可降解连接基团包括但不局限于以下例子，其中X，Y，和R3-R9定义同上：
Figure PCTCN2016110876-appb-000026
在某些实施方式中，Y选自
(1)N-RO；
(2)O，条件是X不是OH或SH。
在某些实施方式中，Y是C(RpRq)，此类可降解连接基团化学式如下，其中X、R3-R9、Rp和Rq定义如上：
Figure PCTCN2016110876-appb-000027
本组可降解连接基团的例子包括但不局限于以下化学式：
Figure PCTCN2016110876-appb-000028
在某些实施方式中，Y是CH2，此类可降解连接基团化学式如下，其中X和R5-R8定义如上：
Figure PCTCN2016110876-appb-000029
在某些实施方式中，此类可降解连接基团化学式如下，其中R5-R8定义如上：
Figure PCTCN2016110876-appb-000030
在某些实施方式中，X是OH或HN-R0。
在某些实施方式中，X是OH。
在某些实施方式中，X是HN-R0。
本组可降解连接基团的例子包括但不局限于以下化学式：
Figure PCTCN2016110876-appb-000031
子类5
在某些实施方式中，U5是C，Y1是N-Rn，Y是C(RpRq)，一类包含6元环的可降解连接基团化学式如下，其中U1-U4，U6，Rn，Rp，Rq和X定义如上：
Figure PCTCN2016110876-appb-000032
在某些实施方式中，此类可降解连接基团包括，但不局限于以下化学式，其中R5-R8，Rn，Rp，Rq，和X定义如上：
Figure PCTCN2016110876-appb-000033
子类6
在某些实施方式中，U5是C，Y1是O，Y是C-RpRq，一类包含6元环的可降解连接基团化学式如下，其中U1-U4，U6，Rp，Rq和X定义如上：
Figure PCTCN2016110876-appb-000034
在某些实施方式中，此类可降解连接基团包括，但不局限于以下化学式，其中R5-R8，Rp，Rq，和X定义如上：
Figure PCTCN2016110876-appb-000035
子类7
在某些实施方式中，Y1是C(O)，C(S)，SO或SO2，本组可降解连接基团化学式如下：
Figure PCTCN2016110876-appb-000036
其中X选自-OH或-HN-R0；
Y选自：
(1)N-RO；
(2)C(RpRq)；
(3)O，条件是X不是OH；
(4)C(RpRq)，当X是HN-R0，其中R0和Rp连同与其相连的原子可以组成4，5或6元杂环；
RO，R0，Rp，Rq和U1-U6的定义如上。
在某些实施方式中，本组可降解连接基团化学式如下，其中R5-R8定义如上：
Figure PCTCN2016110876-appb-000037
某些实施方式中，Y1是C(O)，本组可降解连接基团化学式如下，其中R5-R8定义同上：
Figure PCTCN2016110876-appb-000038
在某些实施方式中，Y是N-RO，其中RO定义同上。
在某些实施方式中，Y是C-RpRq，其中Rp和Rq定义同上。
在某些实施方式中，X是OH或HN-R0，其中R0定义同上。
子类8
在某些实施方式中，J1是C(R10R11)，一类包含6元环的可降解连接基团化学式如下：
Figure PCTCN2016110876-appb-000039
其中U1，U2，U3，U4，U5，U6独立选自CR12和N；
其中X选自-OH或-HN-R0；
Y选自：
(1)N-RO；
(2)C(RpRq)；
(3)O，条件是X不是OH；
(4)C(RpRq)，当X是-HN-R0，其中R0和Rp连同与其相连的原子可以组成4，5或6元杂环；
R0，Ro，Rp，Rq和R10-R12的定义同上。
在某些实施方式中，Y选自N-RO和O(条件是X不是OH)。
在某些实施方式中，Y是C(RpRq)。
在某些实施方式中，此类引入基团化学式e如下，其中R5-R8，Rp，Rq，R10和R11定义同上：
Figure PCTCN2016110876-appb-000040
在某些实施方式中，R10，R11，Rp，Rq是氢原子(H)，此类引入基团化学式如下，其中R5-R8定义同上：
Figure PCTCN2016110876-appb-000041
在某些实施方式中，R10，R11，Rp，Rq是氢原子(H)，此类引入基团化学式如下，其中R5-R8定义同上：
Figure PCTCN2016110876-appb-000042
在某些实施方式中，X是OH或HN-R0，其中R0定义同上。
在某些实施方式中，X是OH。
在某些实施方式中，X是HN-R0其中R0定义同上。
在某些实施方式中，X是NH2。
此类可降解连接基团的例子包括但不局限于以下化学式：
Figure PCTCN2016110876-appb-000043
子类9
在某些实施方式中，一类包括五元环的可降解连接基团化学式如下：
Figure PCTCN2016110876-appb-000044
其中Z1，Z2，Z3，Z4，Z5独立选自CR12，N，NR13，O和S；
X是OH或HN-R0；
Y选自：
(1)N-RO；
(2)C(RpRq)；
(3)O，条件是X不是OH；
(4)C(RpRq)，当X是HN-R0，其中R0和Rp连同与其相连的原子可以组成4、5或6元杂环；
Y1选自
(1)C(R3R4)；
(2)C(O)或-C(S)，条件是Y和A不是C(O)，C(S)，SO或SO2；
(3)O，S，SO，or SO2，条件是Y和A不是O；
(4)N-Rn；和
(5)键；
J1是C(R10R11)或共价键；
R13选自氢原子、低级烷基、低级烷氧基、酰基、酰氧基、烷氧羰基、芳基、苯基、苄基、卤素、卤代甲酰基、卤代低级烷基、三氟甲基、三氟甲氧基、三氯甲基、氰酰、异氰、异氰酸酯(或盐)、异硫氰酸酯、硫氰酸酯(或盐)、低级烷硫基、氨基、亚氨基、氨基低级烷基、低级烷氨基、低级烷氨基、羟基、羟烷基、硝基、腈、异腈、吡啶基、叠氮、羧基、羧酰氨基、乙酸基、硫烷基、碳酸酯(或盐)、氨基甲酸酯、低级烷氨基甲酰、双低级烷氨基甲酰、磺酸基、磺胺、磺酸酯(或盐)、磺酰、亚砜、硫化物、二硫化物和巯基。或者R13选自-SO2-OH，-SO2-NRm1Rm2，-SO2-Rm3，-C(O)Rm8，-C(O)ORm9，-C(O)NRm12Rm13，其中Rm1，Rm2，Rm3，Rm8，Rm9，Rm12，Rm13，独立选自氢原子(H)、(C1-C18)烷基、芳基、(C1-C18烷基)OH、(C1-C18烷基)SH、(C1-C18烷基)COOH、(C1-C18烷基)NH2、(C0-C4烷基)(C5-C6环烷基)、(C0-C10烷基)(C5-C6杂环)、(C0-C4烷基)(C6-C10芳基)、(C0-C4烷基)(C4-C9杂芳基)。
在某些实施方式中，R13包含羟基、氨基、羧基、巯基等功能团，可以与反应性聚合物的交联功能团形成共价键连接。在某些实施方式中，R13包含叠氮化物或炔基，可以通过点击化学与反应性聚合物上对应的炔基或叠氮化物反应。
R0，Ro，Rp，Rq，R3，R4，和R10-R12定义同上。
由Z1-Z5组成的这些五元环的某些例子是四唑和三唑，其中R5定义同上：
Figure PCTCN2016110876-appb-000045
由Z1-Z5组成的这些五元环的某些例子是咪唑，其中R5和R6定义同上：
Figure PCTCN2016110876-appb-000046
由Z1-Z5组成的这些五元环的某些例子是吡咯，其中R5-R7定义同上：
Figure PCTCN2016110876-appb-000047
由Z1-Z5组成的这些五元环的某些例子是吡唑其中R5和R6定义同上：
Figure PCTCN2016110876-appb-000048
由Z1-Z5组成的这些五元环的其它一些例子是呋喃，其中R5和R6定义同上：
Figure PCTCN2016110876-appb-000049
由Z1-Z5组成的这些五元环的一些例子是噻吩，其中R5和R6定义同上：
Figure PCTCN2016110876-appb-000050
由Z1-Z5组成的这些五元环的其它一些例子是噻唑、异噻唑、恶唑和异恶唑，其中R5定义同上：
Figure PCTCN2016110876-appb-000051
在某些实施方式中，Y1是共价键，J1是C(R10R11)，一类包括五元环的可降解连接基团化学式如下：
Figure PCTCN2016110876-appb-000052
在某些实施方式中，J1是C(R10R11)，Y1是共价键，Y是C(RpRq)，一类包括五元环的可降解连接基团化学式如下：
Figure PCTCN2016110876-appb-000053
在某些实施方式中，Y是C(RpRq)，J1是C(R10R11)，一类以三唑为基础的可降解连接基团化学式如下：
Figure PCTCN2016110876-appb-000054
在某些实施方式中，X是OH或HN-R0；
在某些实施方式中，X是OH；
在某些实施方式中，X是HN-R0；
在某些实施方式中，X是NH2。
在某些实施方式中，一类以三唑为基础的可降解连接基团化学式如下，其中R5定义同上：
Figure PCTCN2016110876-appb-000055
在某些实施方式中，Y是C(RpRq)，J1是C(R10R11)，一类以三唑为基础的可降解连接基团化学式如下，其中R5定义同上：
Figure PCTCN2016110876-appb-000056
在某些实施方式中，X是OH或HN-R0；
在某些实施方式中，X是OH；
在某些实施方式中，X是HN-R0；
在某些实施方式中，X是NH2。
在某些实施方式中，Y是C(RpRq)，J1是C(R10R11)，一类以四唑为基础的引入基团化学式如下，其中所有变量定义同上：
Figure PCTCN2016110876-appb-000057
在某些实施方式中，X是OH或HN-R0；
在某些实施方式中，X是OH；
在某些实施方式中，X是HN-R0；
在某些实施方式中，X是NH2。
在某些实施方式中，Y是C(RpRq)，J1是C(R10R11)，一类以咪唑为基础的可降解连接基团化学式如下，其中R5和R6定义同上：
Figure PCTCN2016110876-appb-000058
以咪唑为基础的可降解连接基团的例子包括，但不局限于以下化学式：
Figure PCTCN2016110876-appb-000059
其中m16和m17各自是1-10的整数。
在某些实施方式中，X是OH或HN-R0；
在某些实施方式中，X是OH；
在某些实施方式中，X是HN-R0；
在某些实施方式中，X是NH2。
在某些实施方式中，Y是C(RpRq)，J1是C(R10R11)，一类以吡咯为基础的可降解连接基团化学式如下，其中R5-R7定义同上：
Figure PCTCN2016110876-appb-000060
在某些实施方式中，X是OH或HN-R0；
在某些实施方式中，X是OH；
在某些实施方式中，X是HN-R0；
在某些实施方式中，X是NH2。
在某些实施方式中，Y是C(RpRq)，J1是C(R10R11)，一类以吡唑为基础的可降解连接基团化学式如下，其中R5和R6定义同上：
Figure PCTCN2016110876-appb-000061
在某些实施方式中，X是OH或HN-R0；
在某些实施方式中，X是OH；
在某些实施方式中，X是HN-R0；
在某些实施方式中，X是NH2。
在某些实施方式中，R5和R6各自是氢原子。
在某些实施方式中，Y1和J1是共价键，一类可降解连接基团化学式如下：
Figure PCTCN2016110876-appb-000062
在某些实施方式中，Y是C(RpRq)。
在某些实施方式中，一类以三唑为基础的可降解连接基团化学式如下，其中R5定义同上：
Figure PCTCN2016110876-appb-000063
在某些实施方式中，一类以三唑为基础的可降解连接基团化学式如下，其中R5定义同上：
Figure PCTCN2016110876-appb-000064
在某些实施方式中，一类以三唑为基础的可降解连接基团化学式如下，其中R5定义同上：
Figure PCTCN2016110876-appb-000065
在某些实施方式中，一类以四唑为基础的可降解连接基团化学式如下：
Figure PCTCN2016110876-appb-000066
在某些实施方式中，一类以咪唑为基础的可降解连接基团化学式如下，其中R5和R6定义同上：
Figure PCTCN2016110876-appb-000067
在某些实施方式中，一类以吡咯为基础的可降解连接基团化学式如下，其中R5-R7定义同上：
Figure PCTCN2016110876-appb-000068
在某些实施方式中，一类以吡唑为基础的可降解连接基团化学式如下，其中R5和R6定义同上：
Figure PCTCN2016110876-appb-000069
Figure PCTCN2016110876-appb-000070
在某些实施方式中，X是OH或HN-R0；
在某些实施方式中，X是OH；
在某些实施方式中，X是HN-R0；
在某些实施方式中，X是NH2。
在某些实施方式中，Y1是C(R3R4)，一类可降解连接基团化学式如下：
Figure PCTCN2016110876-appb-000071
在某些实施方式中，Y是C(RpRq)。
在某些实施方式中，一类以三唑为基础的可降解连接基团化学式如下，其中R5定义同上：
Figure PCTCN2016110876-appb-000072
在某些实施方式中，一类以三唑为基础的可降解连接基团化学式如下，其中R5定义同上：
Figure PCTCN2016110876-appb-000073
一类以三唑为基础的可降解连接基团化学式如下，其中R5定义同上：
Figure PCTCN2016110876-appb-000074
在某些实施方式中，一类以四唑为基础的可降解连接基团化学式如下：
Figure PCTCN2016110876-appb-000075
在某些实施方式中，一类以咪唑为基础的可降解连接基团化学式如下，其中R5和R6定义同上：
Figure PCTCN2016110876-appb-000076
在某些实施方式中，一类以吡咯为基础的可降解连接基团化学式如下，其中R6和R7定义同上：
Figure PCTCN2016110876-appb-000077
在某些实施方式中，一类以吡唑为基础的可降解连接基团化学式如下，其中R5和R6定义同上：
Figure PCTCN2016110876-appb-000078
在某些实施方式中，X是OH或HN-R0；
在某些实施方式中，X是OH；
在某些实施方式中，X是HN-R0；
在某些实施方式中，X是NH2。
子类10
在某些实施方式中，Y1是C(O)，一类可降解连接基团化学式如下：
Figure PCTCN2016110876-appb-000079
X是OH或HN-R0；
Y选自：
(1)N-RO；
(2)C(RpRq)；
(3)O，条件是X不是OH；
(4)C(RpRq)，当X是HN-R0，其中R0和Rp连同与其相连的原子可以组成4，5或6元杂环；
其中A，B，Ro，R0，Rp和Rq定义同上。
在某些实施方式中，这类可降解连接基团化学式如下：
Figure PCTCN2016110876-appb-000080
其中a和b定义同上，m和n各自是独立选自0，1，2，3，4，5，和6的整数。
在某些实施方式中，这类可降解连接基团化学式如下，其中R5和R6定义同上：
Figure PCTCN2016110876-appb-000081
在某些实施方式中，Y是C(RpRq)，这类可降解连接基团化学式如下，其中R5和R6定义同上：
Figure PCTCN2016110876-appb-000082
在某些实施方式中，X，Y和C(O)构成一个天然或非天然氨基酸或羟基酸残基。非限制性例子包括丙氨酸、精氨酸、天冬酰胺、天冬氨酸、半胱氨酸、甘氨酸、谷氨酸、 组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、脯氨酸、谷氨酰胺、苯丙氨酸、丝氨酸、苏氨酸、缬氨酸、色氨酸、酪氨酸、氨基异丁酸、肌氨酸、乙醇酸和苯乳酸。
这类可降解连接基团的例子包括但不局限于：
Figure PCTCN2016110876-appb-000083
其中R5和R6定义同上。
这类可降解连接基团的一些更具体的例子包括，但不局限于：
Figure PCTCN2016110876-appb-000084
Figure PCTCN2016110876-appb-000085
在某些实施方式中，X是OH或HN-R0；
在某些实施方式中，X是OH；
在某些实施方式中，X是HN-R0；
在某些实施方式中，X是NH2。
在某些实施方式中，连接在同一个原子，或者连接在被一个键分隔开的两个原子的两个R基团(选自R0、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、RO、Rn、Rp、Rq)和与其连接的原子组成C4-C10环烷基、环烯基、环炔基；饱和或不饱和单环、多环和稠环，可选地被除氢原子(H)之外至少一个基团取代；饱和或不饱和单杂环、多杂环和稠杂环，可选地被除氢原子(H)之外至少一个基团取代；或者两个R基团和与其连接的原子一起组成单芳环或多芳环，可选地被除氢原子(H)之外至少一个基团取代。“至少一个基团”选自低级烷基、低级烷氧基、酰基、酰氧基、烷氧羰基，芳基、苯基、苄基、卤素、卤代甲酰基，卤代低级烷基、三氟甲基、三氟甲氧基、三氯甲基、氰酰、异氰、异氰酸酯(或盐)、异硫氰酸酯、硫氰酸酯(或盐)、低级烷硫基、氨基、亚氨基、氨基低级烷基、低级烷氨基、低级烷氨基、羟基、羟烷基、硝基、腈、异腈、吡啶基、叠氮、羧基、羧酰氨基、乙酸基、硫烷基、碳酸酯(或盐)、氨基甲酸酯、低级烷氨基甲酰、双低级烷氨基甲酰、磺酸基、磺胺、磺酸酯(或盐)、磺酰、亚砜、硫化物、二硫化物和巯基。
在某些实施方式中，R5、R6、R7、R8、R9、R10、R11、R12、R13、Rp、Rq中至少包含一个可以与反应性聚合物形成共价键的反应基团，以此连接至一个或多个反应性聚合物。
根据本发明，所述水凝胶可以通过由骨架部分和可选的交联物组成。
骨架部分的特征是具有带分支的核，从核伸出至少2条链，优选2-16条，更优选4-8条。带分支的核可以由结合形式的多元或低元醇构成，优选季戊四醇、三季戊四 醇、六甘油(hexaglycerin)、山梨糖醇、甘露糖醇、聚乙烯醇、三羟甲基丙烷、蔗糖、果糖、葡萄糖、葡聚糖、纤维素、淀粉、直链淀粉、透明质酸，或者带分支的核可以由结合形式的多元或低元胺构成，例如鸟氨酸、二氨基丁酸、三赖氨酸、四赖氨酸、五赖氨酸、六赖氨酸、七赖氨酸、八赖氨酸、九赖氨酸、十赖氨酸或低聚赖氨酸。
从带分支的核伸出的链可以是任意长度。在一些实施方案中，链原子全部是碳原子。在一些实施方案中，主链的链原子选自C、O、N和S，链原子可以根据它们预期的溶解性进行选择，以便提供溶解度更合适的链。在一些实施方案中，链包含可选的可被酶降解或在体内条件可自动断裂的连接基团。在一些实施方案中，链是由酯键或酰胺键连接在一起的几个片段组成。链可包括但不限于，PEG、长链脂肪酸、天然或非天然氨基酸(例如β-丙氨酸、γ-氨基丁酸、γ-谷氨酸)、短肽(例如β-丙氨酸-β-丙氨酸、γ-谷氨酸-γ-谷氨酸)，或是上述两种或多种的组合(例如，可选的长链脂肪酸、PEG、氨基酸、短肽等通过共价键连接在一起形成的长链)。在一些实施方式中，链是任意长度的肽。示例性的链是从约1至50个氨基酸长度、5至50个、3至5个、5至10个或5至15个氨基酸长度。在某些实施方式中，链是聚赖氨酸、聚谷氨酸、聚天冬氨酸、这些氨基酸的共聚物，及这些氨基酸和其它氨基酸，例如丝氨酸，的混合聚合物。
优选的是PEG基的聚合链。PEG基的聚合链可以是直链的、支链的或分叉的。PEG基的聚合链一端与核连接，另外一端与具有至少一个交联功能团和至少一个可选的反应功能团的结构连接。在某些实施方式中，这种结构具有超枝化部分。聚合链的结构可以相同，也可以不同。每个聚合链上的PEG片段的数量可以不等。PEG基的聚合链中间可以包含烷基、芳基和杂原子。超枝化部分可以提供更多的功能团。在某些实施方式中，超枝化部分的额外功能团可以增加交联物的数量和密度。在某些实施方式中，超枝化部分的额外功能团可以增加药物分子的载量。
带分支的核延伸出来的PEG基的聚合链的优选结构是多臂PEG衍生物，例如商业公司提供的4臂和8臂PEG衍生物。优选地，骨架部分的交联功能团和反应功能团的总和被从带分支的核延伸出的PEG基的聚合链数目均分，从而使每条PEG基的聚合链上有相同数目的交联功能团和反应功能团。
在某些实施方式中，本发明的水凝胶骨架的通式是J(L1-B1)n1，其中J是带分支的核；L1是从中延伸出来的链，两端分别通过共价键连接J和B1。L1可以是直链、支链或分叉结构。在一些实施方案中，链原子全部是碳原子。在一些实施方案中，L1主链的链原子选自C、O、N和S，链原子可以根据它们预期的溶解性进行选择，以便提供溶解度更合适的链。在一些实施方案中，L1包含可选的可被酶降解或在体内条件可自动断裂的连接基团。L1可包括但不限于，PEG、长链脂肪酸、天然或非天然氨基酸(例如β -丙氨酸、γ-氨基丁酸、γ-谷氨酸)、短肽(例如β-丙氨酸-β-丙氨酸、γ-谷氨酸-γ-谷氨酸)，或是上述两种或多种的组合(例如，可选的长链脂肪酸、PEG、氨基酸、短肽等通过共价键连接在一起形成的长链)。优选地，L1是PEG基的聚合链。更优选地，每个L1独立选自式-(CH2)n4(OCH2CH2)nLn5或-(CH2)n4(CH2CH2O)nLn5，其中n4是0-5的整数，n是1-1000的整数，Ln5是连接L1末端和B1的键或者化学功能团。在某些实施方式中，L1是任意长度的肽。在某些实施方式中，L1是聚赖氨酸、聚谷氨酸、聚天冬氨酸、这些氨基酸的共聚物，及这些氨基酸和其它氨基酸，例如丝氨酸，的混合聚合物；B1是具有至少一个交联功能团C2和至少一个可选的反应功能团C1的结构。反应功能团和交联功能团可以相同，也可以不同。同一个骨架上的功能团不能相互反应。n1是2-16的整数。每个L1和B1可以独立选择。在某些实施方式中，B1具有可选的超枝化部分，从而提供多个交联功能团和多个反应功能团。在某些实施方式中，B1具有树枝状聚物结构。优选地，L1和B1通过酰胺键连接。
在一个实施方式中，一个4臂水凝胶骨架的结构如下：
Figure PCTCN2016110876-appb-000086
其中C1是反应功能团，C2是交联功能团。在某些实施方式中，C2与交联物的交联功能团正交反应，C1不能与交联物的交联功能团反应。
在某些实施方式中，水凝胶骨架的通式是(C1-L1)n2J(L1-C2)n3，其中J是带分支的核；L1是从中延伸出来的链；C1是反应功能团，C2是交联功能团。反应功能团和交联功能团可以相同，也可以不同，同一个骨架上的功能团不能相互反应；n2和n3是0-16的整数。
优选地，本发明的水凝胶骨架的通式是(C1-L1)4J(L1-C2)4，其中J是带分支的核，例如六聚甘油和三季戊四醇；L1是从中延伸出来的链；C1是反应功能团；C2是交联功能团。优选地，L1是PEG基的聚合链。更优选地，每个L1独立选自式-(CH2)n4(OCH2CH2)nLn5或-(CH2)n4(CH2CH2O)nLn5，其中n4是0-5的整数，n是1-1000的整数，Ln5是连接L1末端和C1、C2的键或者化学功能团。优选地，L1和C1、C2通过酰胺键连接。每个骨架部分(C1-L1)4J(L1-C2)4的C1和C2可以分别独立选择。在一个实施方式中，8臂水凝胶骨架的结构如下：
Figure PCTCN2016110876-appb-000087
在某些实施方式中，C1和C2可以连接分叉结构或超枝化物，例如下述B1。优选地，B1是高度分支化多肽。在某些实施方式中，所述高度分支化多肽包括一个或多个赖氨酸。一个示范性例子如下：
Figure PCTCN2016110876-appb-000088
波浪线表示与其它部分通过共价键连接。赖氨酸可以是L型或D型。D-赖氨酸能够更好地抵抗酶水解。
一种优选的骨架部分如图所示：
Figure PCTCN2016110876-appb-000089
其中J是带分支的核。每一个分支的赖氨酸残基的数量可以根据需要增加或者减少，赖氨酸的部分氨基可以用于交联，部分可以用于装载生物活性物质。
优选地，所述骨架部分有范围为1-40kDa，更优选5-20kDa的分子量。
交联物是包含至少两个交联功能团和至少一个可选的可生物降解连接基团的化合物。在某些实施方式中，交联功能团通过可生物降解连接基团连接交联物，交联功能团与可生物降解连接基团之间可以根据需要插入用于连接的链。所述交联物的交联功能团能够与水凝胶骨架的交联功能团反应形成共价键。所述交联物的功能团可以相同，也可以不同(异双功能)。所述异双功能交联物的第一个功能团与所述水凝胶骨架的交联功能团反应，第二个功能团与同一个或第二个水凝胶骨架的交联功能团反应。在某些实施方式中，交联物有一个以上的可生物降解连接基团。这些可生物降解连接基团可以相同，也可以不同。同一个交联物上的功能团不能相互反应。
交联物的结构可以是链型，例如C4-Z2-L2-C4、C4-Z2-L2-Z2-C4、C4-Z2-L2-C4′、C4-Z2-L2-Z2-C4′或者C4-Z2-L2-Z2′-C4′，其中L2是连接两端的链，C4和C4′是交联功能团，Z2和Z2′是可选的可生物降解连接基团。C4和Z2、C4′和Z2′之间可以根据需要插入用于连接的链。
交联物的结构可以是多臂结构，比如4臂结构(十字型)、八臂结构等，通式是O(L2-Z2-C4)n6，其中O是带分支的核，L2是从中延伸出来的链，C4是交联功能团，Z2是可选的可生物降解连接基团，n6是2-8的整数。。每一个分支的Z2可以相同，也可以不同。每一个分支的C4可以相同，也可以不同。在一个实施方式中，4臂交联物的结构如下：
Figure PCTCN2016110876-appb-000090
在某些实施方式中，C4不存在，Z2可以直接与骨架部分的交联功能团反应形成共价键。
本发明的水凝胶骨架的交联功能团与交联物的对应位点的交联功能团是关联反应对。一些常用的、非限制性的功能团的关联反应对例子包括：
氨基可以与羧酸、醛和酮反应。
巯基可以参与硫醇-烯反应(Thiol-Ene Reactions)。巯基可以与马来酰亚胺、碘代醋酸、乙烯砜、乙烯璜酰胺、丙烯酰胺、丙烯酸酯、卤代羰基、烯酮(enone)反应。
叠氮化合物可以与炔基(例如有2-20个碳原子的链炔、环辛炔、芳炔)、双环壬炔(bicyclononyne，BCN)、马来酰亚胺反应。点击化学反应中，叠氮-炔基环加成反应包括铜催化的反应和张力促进(strain-promoted)反应。后者常用的功能团包括二苯并环辛炔(dibenzocyclooctyne，DBCO)、单氟环辛炔(monofluorocyclooctyne，MFCO)、二氟化环辛炔(difluorinated cyclooctyne，DIFO)和无芳基环辛炔(aryl-less cyclooctyne，ALO)。不同的环辛炔功能团与叠氮化合物的反应速度可以有1000倍的差异(DBCO＞MFCO＞ALO)。因此，在一个以上的环辛炔功能团存在的情况下，叠氮化合物可以有选择地与其中某个或某几个环辛炔功能团反应，例如先与DBCO反应。另外一类与叠氮化合物通过张力促进反应的是双环壬炔化合物。
硝酮可以与炔基(例如环辛炔)、双环壬炔反应。
狄尔斯-阿尔德环加成反应，例如马来酰亚胺可以与呋喃、1，3-二烯、环戊二烯反应。
四嗪可以与烯烃反应。例如4-(6-甲基-1，2，4，5-四嗪)苯乙酸和降冰片烯通过逆电子需求狄尔斯-阿尔德环加成反应(inverse-electron demand Diels-Alder cycloaddition reaction)反应。另外，四嗪还可以与反式环辛烯(trans-cyclooctene，TCO)反应。
烷氧基胺(alkoxyamine)可以与酮，例如丙酮酰胺(pyruvamide)，缩合生成肟(oxime)。醛可以和羟胺、酰肼反应。
四唑与烯烃光照反应。双硫酯可以与二烯反应。蒽可以与马来酰亚胺反应。
本领域已知的任何化学反应都可以应用于交联，包括酰化、还原烷基化、迈克尔加成、硫醇烷化或通过一个分子上的活性基团(例如醛、氨基、酯、硫醇、α-卤代乙酰、马来酰亚胺、N-羟基琥珀酰亚胺酯或肼基)对同一分子或另外一个分子的活性基团(例如醛、氨基、酯、硫醇、α-卤代乙酰、马来酰亚胺、N-羟基琥珀酰亚胺酯或肼基)化学选择性缀合或者连接。在反应中所用活化基团包括但不限于N-羟基琥珀酰亚胺酯、砜、马来酰亚胺、三氟甲磺酸酯、三氟乙基磺酸酯、以氮丙啶(azidirine)、环氧乙烷和5-吡啶基。
在某些实施方案中，水凝胶骨架部分通过可生物降解键或可生物降解连接基团直接相连，不需要交联物。在某些实施方案中，骨架部分可以通过交联物连接在一起。每个交联物包含至少一个可生物降解的连接基团。所述可生物降解水凝胶可含有一种或多种不同类型的交联物。
在某些实施方式中，交联物是包含至少两个交联功能团、至少一个可生物降解连接基团和至少一个可选的反应功能团的化合物。在某些实施方式中，交联功能团或反应功能团通过可生物降解连接基团连接交联物。交联功能团或反应功能团与可生物降解连接基团之间可以根据需要插入用于连接的链。所述交联物的交联功能团与水凝胶骨架的交联功能团反应形成共价键。所述交联物的交联功能团可以相同，也可以不同(异双功能)。所述异双功能交联物的第一个交联功能团与所述水凝胶骨架的交联功能团反应，第二个交联功能团与同一个或另外一个水凝胶骨架的交联功能团反应。同一个交联物上的功能团不能相互反应。
这种交联物的结构可以是链型。在某些实施方式中，交联物通式是B2-Z2-L2-Z2-B2、B2-Z2-L2-B2、B2-Z2-L2-Z2-B2′或者B2-Z2-L2-Z2′-B2′，其中L2是连接两端的链，B2和B2′分别具有至少一个交联功能团和至少一个可选的反应功能团。在某些实施方式中，B2或B2′具有可选的超枝化部分，从而可以提供多个交联功能团和多个反应功能团。Z2和Z2′是可 选的可生物降解连接基团。B2和B2′、Z2和Z2′可以相同，也可以不同。在一个实施方式中，链型交联物的结构如下：
Figure PCTCN2016110876-appb-000091
其中Z2是可选的可降解连接基团，C4是交联功能团，C6是反应功能团。
B2或B2′的分支结构中可以包含一个或一个以上可选的可生物降解连接基团。在某些实施方式中，链型交联物的结构如下：
Figure PCTCN2016110876-appb-000092
其中Z2、Z3是可选的可降解连接基团，C4是交联功能团，C6是反应功能团。在某些实施方式中，Z2的降解半衰期长于Z3。
在某些实施方式中，交联物通式是B2-L2-B2，或者B2-L2-B2′，其中L2是连接两端的链，B2和B2′具有至少一个交联功能团和至少一个可选的反应功能团，交联功能团和反应功能团可以分别与可选的可降解连接基团相连。同一个交联物上的功能团不能相互反应。在一个实施方式中，链型交联物的结构如下：
Figure PCTCN2016110876-appb-000093
在一个实施方式中，带分支结构(未包括可选的可降解连接基团和与其相连的反应功能团或交联功能团)的PEG基的链型交联物结构如下：
Figure PCTCN2016110876-appb-000094
在某些实施方式中，交联物的通式是O(L2-Z2-B2)n6，其中O是带分支的核，L2是从中延伸出来的链，Z2是可选的可降解连接基团，B2具有至少一个交联功能团和至少一个可选的反应功能团，反应功能团或交联功能团可以与可选的可降解连接基团相连。在某些实施方式中，B2具有可选的超枝化部分，从而可以提供多个交联功能团和多个反应功能团。在某些实施方式中，B2具有树枝状聚物结构。n6是2-8的整数。优选地，L2是PEG基的聚合链，两端分别通过共价键连接带分支的核和Z2。更优选地，每个L2独立选自式(CH2)n4(OCH2CH2)nLn5或(CH2)n4(CH2CH2O)nLn5，其中n4是0-5的整数，n是1-1000的整数，Ln5是连接L2末端和Z2的键或者化学功能团。优选地，L2和Z2通过酰胺键连接。每个O(L2-Z2-B2)4的Z2、B2可以独立选择。
B2的分支结构中可以包含一个或一个以上可选的可生物降解连接基团。在一个实施方式中，4臂交联物的结构如下：
Figure PCTCN2016110876-appb-000095
Z2、Z3、C4和C6的定义同上。在某些实施方式中，Z2的降解半衰期长于Z3。
在某些实施方式中，交联物的通式是O(L2-B2)n6，n6是2-8的整数，B2具有至少一个交联功能团和至少一个可选反应功能团，互联功能团和反应功能团可以分别与可选的可降解连接基团相连。在一个实施方式中，4臂交联物的结构如下：
Figure PCTCN2016110876-appb-000096
在某些实施方式中，C4或C6不存在，Z2、Z3可以直接与骨架部分的功能团反应形成共价键。
在一个实施方式中，带分支结构B2(未包括可选的可降解连接基团和与其相连的反应功能团或交联功能团)的多臂PEG基的交联物结构如下：
Figure PCTCN2016110876-appb-000097
在某些实施方式中，组成聚合交联物部分的单体通过可生物降解的键连接。所述交联物部分的例子包括基于聚羟乙酸或聚乳酸的聚合物。
在一种“4X4”水凝胶中，四臂水凝胶骨架的交联功能团和四臂交联物的交联功能团反应，构成网状结构。优选地，水凝胶骨架和交联物是四臂20kDa PEG基的。一个非限制性的示例如下：
Figure PCTCN2016110876-appb-000098
其中C2是水凝胶骨架的交联功能团，与交联物的对应位点的交联功能团C4是关联反应对。C1是水凝胶骨架的反应功能团，Z2是可生物降解连接基团。
水凝胶中骨架和交联物中包含的反应功能团，可以有多种用途，例如装载生物活性物质或生物标记物。这些反应功能团可以直接使用，可以转化或者衍生为其它正交反应功能团之后使用。
在临床应用中，为了降低或者避免皮下组织对水凝胶的反应，水凝胶的注射量应该尽量小，因此要求水凝胶有高的载药能力。即使水凝胶骨架包含超枝化部分，当药物分子比较大时，空间位阻会影响骨架的装载量。在本发明的某些实施方式中，交联物中采用支链或分叉结构，增加了反应功能团的数量，并利用支链或分叉结构的长度降低了位阻，因此水凝胶的载药潜力显著提高。
本发明中的生物活性物质可以与可降解连接基团连接，而可降解连接基团进而通过可选的反应功能团与所述水凝胶偶联，从而使生物活性物质装载在水凝胶上。在某些实施方式中，本发明中的生物活性物质可以通过其中含氮原子结构(例如氨基)或羟基与可降解连接基团连接，而连接基团进而通过可选的反应功能团与所述水凝胶偶联，连接方式为水凝胶-可选反应功能团-可降解连接基团-生物活性物质。
在某些实施方式中，四臂水凝胶骨架的可选的反应功能团与生物活性物质通过可生物降解连接基团连接。一个非限制性的示例如下：
Figure PCTCN2016110876-appb-000099
其中C2是水凝胶骨架的交联功能团，与交联物的对应位点的交联功能团C4是关联反应对。C1是水凝胶骨架的反应功能团，与反应功能团C3是关联反应对。Z1、Z2是可选的可生物降解连接基团。Drug代表生物活性物质。在某些实施方式中，C3不存在，Z1-drug直接与C1以共价键连接。
在本发明所述水凝胶载体的生物活性物质中，绝大部分生物活性物质的释放(＞90％)最好发生在已经释放显著量的骨架部分(＜10％)之前。这可以通过在骨架之间的交联物和水凝胶与生物活性物质之间的连接链部分插入不同半衰期的可降解连接基团来实现。用于骨架之间的交联物的可降解连接基团的半衰期更长。
在一种“8X4”水凝胶中，八臂水凝胶骨架的4个交联功能团和四臂交联物的4个交联功能团反应，构成网状结构。优选地，水凝胶骨架是八臂40kDa PEG基的，交联物是四臂20kDa PEG基的。一个非限制性的示例如下：
Figure PCTCN2016110876-appb-000100
八臂水凝胶骨架的其余4个反应功能团C1与生物活性物质通过可生物降解连接基团连接。
Figure PCTCN2016110876-appb-000101
其中C1、C2、C3、C4、Z1、Z2和Drug定义同上。
本发明中的水凝胶在体内水解，可以是水凝胶骨架中的可降解键水解，也可以是交联物中的可降解键水解，还可以是水凝胶骨架和交联物都水解。
本发明的可降解连接基团使两个水凝胶骨架单元之间或者水凝胶骨架与交联物之间以酰胺键连接。酰胺键是生物体内最稳定的化学键，以聚乙二醇、聚丙二醇等为基础构建的水凝胶骨架或者交联物不包含人体内酶的典型切割位点。因此，本发明中以此方式构建的水凝胶有很好的体内稳定性，降解速率由可降解基团决定。据发明人了解，这是第一个以酰胺键构建水凝胶并通过酰胺键自动断裂实现水凝胶降解的系统。
采用传统的非共价封装方法的水凝胶一般通过交联的酯键的水解使水凝胶降解，释放生物活性物质。采用共价储存库方法的水凝胶为能够降解，通常在骨架中大量使用酯键，而且生物活性物质往往通过酯键连接到水凝胶。用于聚合物的酯键的水解速 率的变化范围较窄，水凝胶的网络化学和结构在水凝胶的降解速率中起到更大的作用。因此，用于每一种生物活性物质的运送系统需要单独优化。本发明的水凝胶的优势在于使用不需要酶催化的、在生理条件下可降解连接基团。本发明的水凝胶骨架内部可以包含可降解连接基团，或者水凝胶骨架之间通过可降解连接基团连接，或者水凝胶骨架之间通过包含可降解连接基团的交联物连接。水凝胶的网络化学和结构基本不变，从而使生物活性物质的释放和水凝胶的降解速率取决于可降解连接基团的断开动力学，根据给药的要求选择合适降解速率的可降解连接基团即可。本发明提供的可降解连接基团的半衰期有非常广的变化范围，从而满足不同的临床需求。这些水凝胶系统基本不需要为每个生物活性物质单独优化聚合物结构。载体材料的性能，例如生物兼容性(最小刺激性、免疫原性、毒性等)可以在生物活性物质释放性能之外独立优化。
传统药物封装系统在水凝胶降解的初期和末期显示“突释”(burst release)或者后期“剂量倾泻″(dose dumping)，特点是生物活性物质从水凝胶中快速、不可控地释放。早期突释可以占封装的生物活性物质总量的20％。本发明的水凝胶系统，无论是共价储存库方法还是非共价储存库方法，没有同样的问题。
本发明某些实施方式使用非共价封装方法。水凝胶的三维网络中的平均孔径小于生物活性物质的大小，生物活性物质例如药物封装在水凝胶内，没有化学键与水凝胶连接。在某些实施方式中，在生物活性物质存在情况下形成水凝胶可使所述生物活性物质封装在凝胶中。
本发明的某些实施方式使用共价储存库方法。如果装载的生物活性物质分子量是1～100Kda，为使生物活性物质可以穿过水凝胶的孔，水凝胶节点之间的距离不小于7nm。相互交联的PEG基的两个多臂骨架和交联物的核心之间的距离一般不短于10kDa PEG链的长度。如果装载的生物活性物质是小分子化合物，水凝胶节点之间的距离要缩短，可以使用较小分子量的PEG基的多臂骨架和交联物。
本发明的水凝胶可以提供单一生物活性物质，也可以提供一种以上生物活性物质的组合物。组合物中的生物活性物质可以用于治疗同一种疾病，也可以用于治疗不同的疾病。通过选用相同或者不同半衰期的可降解连接基团，不同生物活性物质的释放速率可以相同或者不同。
水凝胶的半衰期是1-10000小时，优选的是1-5000小时，更优选的是1-1000小时。本申请给出的范围，例如1-1000小时，应认为其中间的区间数是被具体而明确地公开，因此不必列举一长串的数值。本申请包括在此范围内的任何随机范围，例如1-1000包括1-500和100-500。
生物药例如多肽或蛋白在体内作用时间短的一个重要因素是体内酶降解，另外一个是肾清除。延长药物体内作用时间的常用方法是使多肽或蛋白与天然或合成大分子通过可水解或不可水解键连接。生物大分子包括白蛋白、多糖(如葡聚糖)、抗体(如IgG或者IgG Fc)等。合成大分子包括聚乙二醇等。药物与大分子通过不可水解共价键连接后，生物活性往往会大幅度降低。药物与大分子通过可水解键(如酯，碳酸，可水解氨基甲酸酯)结合，目前常用的可水解键稳定性差，在血浆中易于被酶降解或自发水解，从而使药物释放难以预测。另外，很多生物活性物质，尤其是分子量较小的药物，缺少能够与大分子缀合的合适功能团。缀合法或者融合蛋白法都只能使酶降解和肾清除降低到一定程度。PEG40K是目前临床上使用的最大分子量聚乙二醇，但即使增加聚乙二醇分子量到60K或80K，延长的时间很有限，而且由于聚乙二醇分子量太大，增加了毒副作用的风险。所以依赖大分子的长效方法有难以逾越的时间上限。
本发明的水凝胶以皮下组织为药物储存库，在药物释放之前，没有酶降解和肾清除的问题，因此给药时间可以延长到周、月甚至年。现有长效药物的给药方式是将多次，甚至多天的剂量一次投入，初期血药浓度很高，增加了治疗指数低的药物的毒副作用。虽然设计没有生物活性的前药是一种解决方案，但很多生物活性物质的活性位点没有适于缀合的功能团，而大分子与远端的功能团缀合，不一定能抑制其活性。本发明的水凝胶在生物活性物质释放前，将其限制在注射位点，无法到达靶点，因此不显示生理活性，效果类似于前药。不像前药，本发明的水凝胶可以适应生物活性物质的各种功能团类型和位置。水凝胶缓慢释放药物，血药浓度曲线平稳，提高了治疗指数低的药物的安全性，改善了药物的治疗效果。另外，蛋白药物通常含有多个半胱氨酸或者赖氨酸，定点缀合难度大，经常无法得到单一产品。多数聚乙二醇化蛋白药物都存在这个问题。例如，在
Figure PCTCN2016110876-appb-000102
(牛pegademase)和
Figure PCTCN2016110876-appb-000103
(pegaspargase)中，聚乙二醇的可能连接位点包括赖氨酸、丝氨酸、酪氨酸和组氨酸等。猪尿酸酶是一个四聚体，每个子单元包含28-29个赖氨酸理论上可以与聚乙二醇反应，实际上每个子单元能够与平均10-11个PEG10KDa缀合。聚乙二醇尿酸酶(
Figure PCTCN2016110876-appb-000104
pegloticase)中尿酸酶的每个子单元包含平均9个聚乙二醇分子。INF-α2a的8个赖氨酸可以与聚乙二醇反应生成8个不同的产物，其中活性最高的产物与活性最低的产物的活性有3倍差距(Foser等，蛋白表达和纯化2003，30，78-87)。本发明的非共价封装方法并不涉及与药物进行反应，药物在水凝胶中和释放后都是原始序列和结构。本发明的共价储存库方法与药物通过共价键连接，连接位点可能无法做到一样，但药物释放后仍然是原始序列和结构，保证了实际有效药物的单一性。
水凝胶的制备
本发明提供用于制备可生物降解水凝胶的方法，所述方法包括水凝胶骨架与交联物反应，其中交联物包含可以与骨架对应位点反应的功能团和可生物降解连接基团。
在某些实施方式中，可生物降解水凝胶通过使同一种骨架与可降解交联物反应来制备。所述骨架是多价的，因此可以在所述三维水凝胶基体中形成节点。一个例子就是多臂PEG与交联剂反应生成水凝胶。具有不同反应功能团和分子量的多臂PEG试剂市售可得。另外一个例子，使直链聚合物与交联物反应形成水凝胶。直链聚合物的例子包括透明质酸、聚乙烯醇、羧甲基纤维素、聚(2-羟乙基甲基丙烯酸酯)、葡聚糖、壳聚糖、胶原、藻酸盐和琼脂糖。
在某些实施方式中，本发明提供用于通过一种以上的不同的骨架和可降解交联物反应生成可生物降解水凝胶的方法。可降解交联物包含与所述第一个骨架反应的功能团1、与第二个骨架反应的功能团2和可降解连接基团。功能团1和2可以相同或不同。优选地，骨架包含至少2臂，更优选是至少4臂。
用于本发明的反应性聚合物可以是均聚或共聚的聚乙二醇、聚丙二醇、聚(N-乙烯吡咯烷酮)、聚甲基丙烯酸酯、聚磷腈、聚交酯、聚丙烯酰胺、聚乙醇酸、聚乙烯亚胺、琼脂糖、葡聚糖、明胶、胶原、聚赖氨酸、壳聚糖、藻酸盐、透明质酸、果胶、角叉聚糖或聚氨基酸，其在天然状态下具有合适的反应功能团和交联功能团，或经衍生从而具有合适的反应功能团和交联功能团。合适的反应功能团和交联功能团包括但不限于：胺类、醇类、硫醇类、羧酸类、马来酰亚胺、丙烯酸酯类、丙烯酰胺类、叠氮化物类、炔烃类(包括环炔烃类)、1，3-二烯类(包括环戊二烯类和呋喃类)、α-卤代羰基类、N-羟基琥珀酰亚胺类、N-羟基磺基琥珀酰亚胺酯类或碳酸酯类。
本发明的水凝胶还可以包含凝胶化过程中未消耗的反应功能团，这些功能团因为过量或者正交反应性没有参与凝胶化过程。这些功能团可用于进一步修饰所述水凝胶，例如通过共价键连接生物活性物质。
释药水凝胶的制备方式主要有三种。在某些实施方式中，骨架上的反应功能团先与反应功能团-可选可降解连接基团-生物活性物质反应，得到中间体载药聚合物。然后骨架上的交联功能团与交联物反应生成载药的可降解水凝胶。在某些实施方式中，骨架上的交联功能团先与交联物反应生成水凝胶，然后骨架上的反应功能团与反应功能团-可选可降解连接基团-生物活性物质反应，生成载药的可降解水凝胶。在某些实施方式中，骨架、交联物和反应功能团-可选可降解连接基团-生物活性物质在单一步骤中合并(一锅法)。如果在载药水凝胶生成后仍然有未反应的功能团，可以选用适当的试剂对所述过量的功能团加帽。
通过选择不同结构和尺寸的骨架和交联物可以制备微孔、中孔或大孔水凝胶。微孔水凝胶是指具有直径小于1nm的孔的水凝胶，中孔水凝胶是具有直径1-100nm的孔的水凝胶，大孔水凝胶是具有直径大于100nm的孔的水凝胶。
本发明的水凝胶可以在体外制备，然后植入体内。所述水凝胶可以制成特定的形状。在某些实施方式中，所述水凝胶为成型的制品例如支架或网。
本发明的水凝胶可以按照微粒形式通过制备方法获得。优选地，所述水凝胶是珠状微粒，可以通过注射器皮下注射或者肌肉注射。珠状微粒的直径1-500微米。优选地，如果悬浮在等渗水制剂缓冲液中，所述微粒直径10-100微米，优选20-100微米，最优选30-70微米。优选地，微粒可使用内径小于0.6mm的注射针头注射，优选内径小于0.3mm的注射针头，更优选内径小于0.25mm的注射针头，甚至更优选用内径小于0.175mm的注射针头，最优选用内径小于0.16mm的注射针头。
或者，所述水凝胶可通过原位凝胶化来形成。先制备药学上可接受的水凝胶制剂成份，混合所述成份，然后在凝胶化之前注射或应用。注射可以是皮下、肌内、眼内、瘤内或静脉注射。本发明的水凝胶可局部应用，例如向皮肤或手术创口使用混合成份，然后原位凝胶化。本发明的水凝胶还有其它的应用，例如再生医学、骨科或矫形科的植入物、医疗器械的涂层或外科敷料。
本发明通过以下实施例进一步说明，但不限于这些实施例。
具体实施方式
保护基：
Bom，benzyloxymethyl：苄氧甲基；Br-Z，2-bromobenzyloxycarbonyl：2-溴苄氧羰基；tBu，t-butyl：叔丁基；Bz，benzoyl：苯甲酰基；Bzl，benzyl：苄基；Boc：叔丁氧羰基；CHO formyl：甲酰基；cHx，cyclohexyl：环己基；Cbz或Z benzyloxycarbonyl：苄氧羰基；Cl-Z，2-chlorobenzyloxycarbonyl：2-氯苄氧羰基；Fm，9-fluorenylmethyl：9-芴基甲基；Fmoc，9-fluorenylmethoxycarbonyl：9-芴甲氧羰基；Mtt，4-methyltrityl：4-甲基三苯甲基；Pmc，(2，2，5，7，8-pentametylchroman-6-sulphonyl：2，2，5，7，8-五甲基-6-羟基色满；Tos，4-toluenesulphonyl：对甲苯磺酰；Trt，tripheylmethyl：三苯甲基。
试剂和溶剂：
ACN，acetonitrile：乙腈；BOP，benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate：苯并三唑-1-三(三甲氨基)-六氟磷酸酯(卡特缩合剂)；DCC，N，N′-Dicyclohexylcarbodiimide：二环己基碳化二亚胺；DCM：二氯甲烷；DEPBT，3- (Diethoxyphosphoryloxy)-1，2，3-benzotriazin-4(3H)-one：3-(二乙氧基邻酰氧基)-1，2，3-苯并三嗪-4-酮；DIC，N，N′-Diisopropylcarbodiimide：N，N′-二异丙基碳二亚胺；DIPEA(或DIEA)，diisopropylethylamine：二异丙基乙胺；DMAP，4-N，N-dimethyl amino pyridine：4-N，N二甲氨基吡啶；DMF：N，N-二甲基甲酰胺；DMSO：二甲亚砜；EDC或EDCl，1-ethyl-3-(3-dimethylaminopropyl)carbodiimide：1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐；EtOAc：乙酸乙酯；HBTU O-(1H-benzotriazole-1-yl)-N，N，N′，N′-tetramethyluroniumhexafluorophosphate：苯并三氮唑-N，N，N′，N′-四甲基脲六氟磷酸盐；HOBT 1-hydroxybenzotriazole：1-羟基-苯并-三氮唑；Cl-HOBT：6-氯-1-羟基-苯并-三氮唑；MTBE，methyl tert-butyl ether：甲基叔丁基醚；NMM，N-Methylmorpholine：N-甲基吗啉；NMP，N-methylpyrrolidinone：N-甲基吡咯烷酮；Piperidine：哌啶；Su，succinimide：琥珀酰亚胺；TEA，triethylamine：三乙胺；TFA，trifluoroacetic acid三氟乙酸；THF，tetrahydrofuran四氢呋喃；TIS，triisopropylsilane：三异丙基硅烷
PEG20K 4臂和PEG40K 8臂试剂购自NOF America。
若无特殊说明，本发明中使用的试剂均来自商业渠道，或者是本领域常规配制试剂，所述实验及其步骤均是本领域技术人员根据本发明内容和本领域常规技术可以完成的。
多肽化学合成方法
线性多肽使用Boc固相多肽合成法或Fmoc固相多肽合成法。如果使用Fmoc化学合成C-末端是羧基的多肽，选用Wang树脂；C-末端是酰胺的多肽选用Rink amide树脂。如果使用Boc化学合成C-末端是羧基的多肽，选用Pam树脂；C-末端是酰胺的多肽选用MBHA树脂。缩合剂和活化剂是DIC和HOBT，其它可选肽键缩合剂包括EDC、BOP、HBTU、DEPBT等。氨基酸5倍过量。缩合时间为1小时。
Fmoc保护基用50％哌啶/DMF脱除。Boc保护基用TFA脱除。肽键缩合反应用茚三酮试剂监测。
使用Fmoc固相多肽合成法时，通用氨基酸及保护基如下：
Fmoc-Cys(Trt)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-His(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Arg(Pmc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Boc-Trp(Boc)-OH或Fmoc-Tyr(tBu)-OH。
固相Fmoc化学合成多肽后，常用的切割试剂是TFA。将干树脂放入摇瓶中，加入适当量三氟乙酸/1，2-乙二硫醇/水(95∶2.5∶2.5，10-25mL/g树脂)，震荡2小时后抽滤树脂，向滤液加入8-10倍体积的冰乙醚。最后，离心收集沉淀出来的多肽粗品。
使用Boc固相多肽合成法时，通用氨基酸和保护基如下：
Boc-Cys(4-MeBzl)-OH、Boc-Asp(OcHx)-OH、Boc-Glu(OcHx)-OH、Boc-His(Bom)-OH、Boc-Lys(2-Cl-Z)-OH，Boc-Asn(Xan)-OH、Boc-Arg(Tos)-OH、Boc-Ser(Bzl)-OH、Boc-Thr(Bzl)-OH、Boc-Trp(CHO)-OH和Boc-Tyr(2-Br-Z)-OH。
固相Boc化学合成多肽后，PAM和MBHA树脂一般用HF切割，每0.1毫摩尔树脂加5毫升HF，另外加入对甲苯酚、对巯基苯酚或苯甲醚，混合物在冰浴条件下搅拌1小时。HF真空抽干后，多肽用冰乙醚沉淀，离心收集沉淀的多肽粗品。
实施例1
Figure PCTCN2016110876-appb-000105
Boc-Lys(Fmoc)-OH(187mg，400μmole)，DEPBT(120mg，400μmole)，DIPEA(140μL，800μmole)加入PEG20K-(NH2)4(2g，100μmole)的二氯甲烷溶液(30mL)，反应在室温搅拌4小时。向反应中加入醋酸酐(50μL，526μmole)，继续搅拌15分钟。反应溶液浓缩到10mL，加入MTBE(100mL)搅拌15分钟，固体沉淀过滤，真空干燥。两批反应产物合并，直接用于下一步反应。
[Boc-Lys(Fmoc)-NH]4-PEG20K(4.14g，190μmole)溶于DMF(25mL)，加入哌啶(2mL，20mmol)。反应在室温搅拌20分钟后，加入MTBE(150mL)，过滤后得到产物[Boc-Lys-NH]4-PEG20K(3.14g，79％)。
DBCO-OSu(81.2mg，200μmole)和DIPEA(69μL，400μmole)加入[Boc-Lys-NH]4-PEG20K(837mg，40μmole)的乙腈溶液(8mL)。反应在室温搅拌2小时，然后加入醋酸酐(20μL，210.4μmole)，继续搅拌20分钟。减压除去溶剂后，固体用HPLC纯化得到[Boc-Lys(DBCO)-NH]4-PEG20K(776mg)。
[Boc-Lys(DBCO)-NH]4-PEG20K(776mg)溶于CH2Cl2(2ml)，加入TFA(1.5ml)。反应在室温搅拌10分钟，加入乙醚(50mL)，沉淀过滤后真空干燥，得到白色粉末[H-Lys(DBCO)-NH]4-PEG20K(653mg)。
实施例2
PEG40K(NH2)8(2g，50μmole)溶于乙腈(50mL)，加入Fmoc-OSu(67.5mg，200μmole)和DIEA(35μl，200μmole)，室温搅拌过夜。减压蒸发乙腈到10mL。两批反应的产物合并，加入含0.1％TFA的水溶液，用制备HPLC纯化，A溶液：0.1％TFA B溶液：0.1％TFA，100％乙腈，得到PEG40K(NH-Fmoc)4(NH2)4(0.83g)。其它的不正确产物可以脱除Fmoc，重新反应循环使用。
PEG40K(NH-Fmoc)4(NH2)4(0.8g)溶于乙腈(5mL)，加入DBCO-OSu(36.2mg)和DIEA(35μL)，室温搅拌3小时。反应用透析方法纯化(Slide-A-lyzer Dialysis Cassette，20K MWCO，赛默飞世尔科技)，得到PEG40K(NH-Fmoc)4(DBCO)4(0.71g)。
PEG40K(NH-Fmoc)4(DBCO)4(0.71g)溶于DMF(6ml)，加入哌啶(173μl)，反应在室温搅拌15分钟。向反应中滴加MTBE，沉淀过滤，用MTBE洗涤3遍，真空干燥得到白色固体PEG40K(NH2)4(DBCO)4(0.6g)。
碘醋酸(11.2mg，60μmole)，DEPBT(18mg，60μmole)和DIEA(10.4μL，60μmole)加入PEG40K(NH2)4(DBCO)4(0.6g)的二氯甲烷溶液(3mL)，室温搅拌8小时，向反应中滴加MTBE，沉淀过滤，用MTBE洗涤3遍，真空干燥得到白色固体PEG40K(NHC(O)CH2l)4(DBCO)4(0.6g)。
实施例3
Figure PCTCN2016110876-appb-000106
向化合物1A(21.0g，119mmol)的CCl4(200mL)溶液加入NBS(23.5g，132mmol)和BPO(2.90g，12.0mmol)。化合物加热到70℃搅拌16小时。混合物用正己烷稀释，过滤沉淀，滤饼用正己烷洗。滤液真空浓缩得到化合物1B(36.0g，粗品)是黄色的油，不纯化直接用于下一步反应。
向化合物1B(26.0g，102mmol)和二亚氨基二羧酸二叔丁酯(22.2g，102mmol)的2-丁酮溶液(250mL)加入Cs2CO3(33.3g，102mmol)。混合物加热到80℃，搅拌16小时。混合物过滤，滤液真空浓缩得到化合物1C(44.0g，粗品)是棕色油，不纯化直接用于下一步反应。
向化合物1C(44.0g，112mmol)的乙酸乙酯溶液(200mL)加入HCl/EtOAc(4M，220mL)，混合物搅拌16小时。混合物过滤，滤饼用乙酸乙酯洗涤。剩余物真空干燥得到化合物1D(12.5g，55.1mmol，49％产率)是白色固体。
1H NMR：400MHz MeODδ8.27(d，1H，J＝7.6Hz)，7.96-7.99(m，2H)，4.46(s，2H)，4.01(s，3H).
向化合物1D(17.5g，77.2mmol)的甲醇溶液(200mL)加入TEA(15.6g，154mmol，21.41mL)，混合物搅拌16小时。混合物真空浓缩，残余物用乙酸乙酯(100mL)和水(200mL)稀释。水层用乙酸乙酯萃取(100mL x 2)，合并的有机层用饱和食盐水(100mL)洗，用无水硫酸钠干燥，真空浓缩得到化合物1E(13.0g，粗品)是黄色油，不纯化直接用于下一步反应。LC-MS：[M+H]+＝158.8
向化合物1E(13.0g，82.2mmol)的二氯甲烷溶液(100mL)加入Boc2O(21.5g，98.6mmol，22.6mL)和DMAP(10.0g，82.2mmol)。混合物搅拌30分钟。混合物用二氯甲烷(100mL)和水(200mL)稀释。水层用二氯甲烷萃取(100mL x 2)，合并的有机层用饱和食盐水(100mL)洗，用无水硫酸钠干燥，真空浓缩得到化合物1F(27.0g，粗品)是黄色油，不纯化直接用于下一步反应。LC-MS：[M+Na]+＝280.9
向化合物1F(27.0g，104mmol)的THF(200mL)和水(200mL)溶液加入氢氧化钠(12.5g，313mmol)。化合物搅拌1小时。混合物中加入水(200mL)，水层用10％柠檬酸中和(300mL)至pH 2～3，用乙酸乙酯萃取(200mL x 2)。合并的有机层用饱和食盐水(200mL)洗，用无水硫酸钠干燥，真空浓缩得到化合物1G(12.0g，43.4mmol，41％产率)是白色固体。
1H NMR：400MHz CDCl3δ8.27(d，1H，J＝7.6Hz)，8.07(t，1H，J＝6.8Hz)，7.66-7.70(m，2H)，4.59(d，1H，J＝6.8Hz)，1.48(s，9H).
向化合物1G(12.0g，43.4mmol)的含氨甲醇溶液(150mL)加入Raney-Ni(12.0g，140mmol)。混合物在50Psi的氢气环境搅拌16小时。混合物用硅藻土过滤，滤饼用甲醇(200mL)洗。滤液真空浓缩得到化合物1I(8.00g，粗品)是白色固体1H，不纯化直接用于下一步反应。LC-MS：[M-99]+＝180.9
向化合物1H(8.00g，28.5mmol)的二恶烷(50mL)和水(50mL)溶液加入碳酸氢钠(7.19g，85.6mmol，3.33mL)。搅拌10分钟后加入Fmoc-Cl(7.38g，28.5mmol)。混合物搅拌16小时。混合物用乙酸乙酯(100mL)和水(100mL)稀释。水层用乙酸乙酯萃取(100mL x 2)。合并的有机层用饱和食盐水(100mL)洗，用无水硫酸钠干燥，真空浓缩，残余物用硅胶柱纯化。洗脱液真空浓缩得到目标化合物1I(2.00g，3.98mmol，14％产率)是白色固体。LC-MS：[M-99]+＝403.0
1H NMR：400MHz DMSO-d6δ12.9(s，1H)，7.86-7.89(m，3H)，7.80-7.84(m，1H)，7.66-7.68(m，2H)，7.37-7.39(m，2H)，7.30-7.32(m，3H)，7.13-7.25(m，2H)，4.43(d，2H，J＝6.4Hz)，4.30(d，2H，J＝7.2Hz)，4.18-4.21(m，3H)，1.35(s，9H)。
实施例4
Figure PCTCN2016110876-appb-000107
化合物1I(1g，2mmole)，DEPBT(598mg，2mmole)，DIPEA(348μL，2mmole)逐次加入PEG20K-(NH2)4(10g，500μmole)的二氯甲烷溶液(30mL)，反应在室温搅拌4小时。向反应中加入醋酸酐(50μL，526μmole)，继续搅拌15分钟。反应溶液浓缩到10mL，加入MTBE(100mL)搅拌15分钟，固体沉淀过滤，真空干燥。中间产物(10.5g，477μmole)溶于DMF(50mL)，加入哌啶(10ml，100mmol)。反应在室温搅拌20分钟后，加入MTBE(500mL)，过滤后得到产物2B，直接用于下一步反应。
向化合物2B(9g，429μmole)的二氯甲烷溶液(40mL)加入5-叠氮戊酸(64mg，56.5μL，450μmole)，DEPBT(135mg，450μmole)，DIEA(58mg，78μL，450μmole)。反应室温搅拌12小时，反应溶液浓缩到15mL，加入MTBE(100ml)搅拌15分钟，过滤固体沉淀，用Sephadex G-50纯化，真空干燥，得到白色粉末2C(8.2g)。
实施例5
Figure PCTCN2016110876-appb-000108
[H-Lys(DBCO)-NH]4-PEG20K(50mg)和2C(50mg)先分别溶于PBS(2mL)，混合后静置5小时，得到含有自由氨基的水凝胶，其骨架与交联物之间的连接方式如3A所示。水凝胶连续用纯净水(2mL)、乙醇(2mL)和乙腈(2mL)洗，真空干燥。水凝胶的自由氨基可以继续衍生为其它正交反应功能团，用于不同的目的，比如装载生物活性物质。可降解连 接基团上的Boc保护基可以在后续的合成的过程中用TFA切割。凝胶化时间定义为溶液从液体转变为胶体，不再能够用移液枪提取。3A的凝胶化时间是2分钟。
实施例6
Figure PCTCN2016110876-appb-000109
实施例5中得到的水凝胶(50mg)悬浮于乙腈(200μL)，加入DBCO-OSu(4mg，10μmol)和DIEA(2μL，11.5μmol)。12小时后，除去上清液，水凝胶用乙腈(2X1mL)洗。重新悬浮于乙腈(200μL)，加入DIEA(4μL，23μmol)和醋酸酐(2.2μL，23μmol)，1小时后除去上清液并干燥。向水凝胶加入50％TFA/CH2Cl2(200μL)，15分钟后除去。水凝胶用乙腈(2X1mL)洗，真空干燥。这种DBCO取代的水凝胶骨架与交联物之间的连接方式如3B所示，可以与N3-可降解连接基团-生物活性物质反应，从而成为给药型水凝胶。
实施例7
Figure PCTCN2016110876-appb-000110
实施例5中得到的水凝胶(50mg)悬浮于DMF(500μL)，加入MAL-dPEG4-NHS(Quanta Biodesign，5.3mg，20μmol)和DIEA(4μL，23μmol)。30分钟后除去上清液，连续用纯净水(1mL)、乙醇(1mL)和乙腈(1mL)洗，真空干燥，得到水凝胶，其骨架与交联物之间的连接方式如3C所示。。
实施例8
Figure PCTCN2016110876-appb-000111
向2B(2g)的DMF溶液(40ml)加入Fmoc-L-谷氨酸-γ-2-苯基异丙酯(CAS 200616-39-3)(53.6mg，110μmole)、DEPBT(33mg，110μmole)和DIEA(19μL，110μmole)，室温搅拌过 夜。减压蒸发溶剂后，固体残留溶于1％TFA/CH2Cl2(10ml)，室温搅拌10分钟，减压蒸发溶剂后，用HPLC纯化，得到[Fmoc-Glu-NH]4-2B(1.87g)。
[Fmoc-Glu-NH]4-2B(1g)溶解于DMF(15mL)，加入N，N’-二环己基碳二亚胺(41.7mg，200μmole)和N-羟基琥珀酰亚胺(23.4mg，200μmole)。反应在室温搅拌过夜，过滤沉淀后，减压蒸发溶剂，得到白色固体4C(0.7g)。
Figure PCTCN2016110876-appb-000112
[H-Lys(DBCO)-NH]4-PEG20K(0.5g)和4C(0.5g)溶于DMSO(2mL)，加入Arlacel P135(20mg)的庚烷溶液(20mL)。混合物快速搅拌，滴加N，N，N′，N′-四甲基-乙二胺(0.2mL)。继续室温搅拌15小时。加入醋酸(0.5mL)和水(15mL)。搅拌5分钟后将水相分离。将水-水凝胶混悬液在200、300、360和500目钢筛上湿筛，分别收集，连续用水、乙醇和乙腈洗，然后悬浮于DMF(5mL)，加入哌啶(1mL)，15分钟后倒掉上清液，用二氯甲烷、水和乙醇洗，真空干燥，得到白色粉末状水凝胶珠子，其骨架与交联物的连接方式如4D，其中自由氨基可以继续衍生为其它正交反应功能团。这种水凝胶包含两种不同的反应功能团，可以用于不同的目的，比如装载两种不同的生物活性物质。可降解连接基团上的Boc保护基可以在后续合成的过程中用TFA切割。与3C相比，在交联物中增加反应功能团可以提高药物装载量。
实施例9
4-((3-(((9H-芴-9-基)甲氧基)羰基)氨基)丙基)氨基甲酰基)-2-(((叔丁氧羰基)氨基)甲基)苯甲酸的合成
Figure PCTCN2016110876-appb-000113
向6A(10g，57.4mmol)二氯甲烷溶液(200mL)加入饱和碳酸氢钠(200mL)和FmocCl(17g，66mmol)。混合物搅拌过夜。混合物分离，有机层用水、饱和食盐水洗，无水硫酸钠干燥，真空浓缩。残余物用快速柱色谱法纯化得到6B(15g，66％).
向6B(15g，38mmol)二氯甲烷溶液(200mL)加入TFA(100mL)。混合物在室温搅拌3小时。混合物真空浓缩。残余物倒入冰水(200mL)，用碳酸氢钠溶液碱化，使pH＞8。混合物过滤，滤饼用水洗，真空干燥得到6C(11g，98％)是白色固体。
向6D(50g，0.22mol)CCl4溶液(750mL)加入N-溴代琥珀酰亚胺(38.9g，0.22mol)和过氧苯甲酰(1.1g，4.4mmol)。反应混合物在250W灯照下在60℃搅拌4小时。混合物冷却到室温，用1M氢氧化钠水溶液和水洗，用无水硫酸钠干燥。蒸发溶剂得到6E粗品(75g)是油，直接用于下一步反应。
6E粗品(75g)悬浮于NH3/MeOH(2M，750mL)和浓氢氧化铵(250mL)搅拌过夜。混合物过滤，滤饼真空干燥得到6F(35g，两步75％产率)是白色固体。
1H-NMR(400MHz，DMSO_d6)：8.64-8.66(s，1H)，7.83(s，1H)，7.66-7.68(d，1H)，7.59-7.61(s，1H)，4.37(s，2H)
向6F(6g，28mmol)甲醇溶液(100mL)加入醋酸钯(0.12g)，Et3N(8.6g，85mmol)和4，5-双二苯基膦-9，9-二甲基氧杂蒽(0.6g)。反应混合物在140℃的CO(3MPa)环境下搅拌过夜。混合物冷却到室温过滤。滤液真空浓缩得到6G(6g).
1H-NMR(400MHz，DMSO_d6)：8.82-8.84(s，1H)，8.16-8.18(s，1H)，8.05-8.07(s，1H)，7.79-7.81(s，1H)，4.44-4.46(s，2H)，3.90-3.92(s，3H)
向6G(6g)CH2Cl2溶液(180mL)二碳酸二叔丁酯(12.2g，56mmol)，Et3N(2.8g，28mmol)和DMAP(3.4g，28mmol)。反应混合物室温搅拌2小时。混合物用0.5N HCl和水洗，用无水硫酸钠干燥，真空浓缩。粗品用快速柱色谱法纯化得到6H(4.2g，51％两步反应)是白色固体。
1H-NMR(400MHz，CDCl3)：8.15-8.17(m，2H)，7.96-7.98(m，1H)，4.80-4.82(s，2H)，3.96-3.98(s，3H)，1.59-1.61(s，9H)
向6H(5.8g，20mmol)的THF溶液(100mL)加入LiOH.H2O(4.2g，100mmol)水溶液(100mL)。反应混合物室温搅拌5小时。反应混合物真空浓缩。水层用柠檬酸酸化到PH＜6，然后过滤。滤饼真空干燥得到6I(4.6g，78％)是白色固体。
1H-NMR(400MHz，DMSO_d6)：7.86-7.98(m，3H)，7.38-7.40(m，1H)，4.48-4.49(m，2H)，1.40-1.42(s，9H)
向6I(3g，10.1mmol)和6C(3g，10.1mmol)的DMF溶液(30mL)加入EDCl(1.9g，10.1mmol)、HOBt(1.3g，10.1mmol)和NMM(1.11mL，10.1mmol)。混合物室温搅拌2小时。三批合并，倒入水中，用乙酸乙酯萃取。合并的有机层用饱和食盐水洗，用无水硫酸钠干燥，过滤，真空浓缩。残余物用快速柱色谱法纯化得到粗品(1.7g)。粗品重结晶得到纯品6J(1.1g，6.3％)是白色固体。
1H-NMR(400MHz，DMSO_d6)：13.21(br，1H)，8.55-8.53(m，1H)，7.89-7.87(m，4H)，7.76-7.67(m，3H)，7.43-7.23(m，6H)，4.50-4.20(m，5H)，3.32-3.24(m，2H)，3.07-3.02(m，2H)，1.70-1.65(m，2H)，1.40-1.27(m，9H).LC-MS：m/z＝473.9(M-100+1).
实施例10
Figure PCTCN2016110876-appb-000114
向7A(5.0g，14.3mmol)的二氯甲烷溶液(100mL)加入三乙胺(2.9g，28.7mmol，3.98mL)、HATU(6.55g，17.2mmol)和N-羟基琥珀酰亚胺(1.98g，17.2mmol)。反应搅拌2小时，得到7B，不纯化直接用于下一步反应。
向7B(1.28g，2.87mmol)的二氯甲烷溶液(20mL)加入1，3-丙二胺(5.00g，28.7mmol)的二氯甲烷溶液(40mL)，室温搅拌12小时。减压蒸发溶剂，残余物用硅胶层析柱纯化得到7C(500mg，43％产率)是黄色固体。
1H NMR：400MHz CDCl3δ7.41-7.39(m，6H)，7.28-7.26(m，6H)，7.24-7.17(m，3H)，6.30(br，s，1H)，3.25(q，J＝6.0Hz 2H)，2.75(t，J＝6.0Hz，2H)，2.49-2.38(m，5H)，2.05(t，J＝6.8Hz，2H)，1.62-1.59(m，2H).LCMS：(M+23)+：427.2.
向6I(729mg，2.47mmol)的二氯甲烷溶液(20mL)加入三乙胺(1.00g，9.88mmol，1.37mL)、HATU(939mg，2.47mmol)和7C(1.00g，2.47mmol)。反应搅拌12小时。反应混合物用二氯甲烷(20mL)和水(20mL)稀释。水层用二氯甲烷萃取。有机层合并用饱和食盐水(20mL)洗，无水硫酸钠干燥，减压浓缩得到残余物。残余物用制备HPLC纯化得到7D(1.00g，59％产率)是白色固体。
1H NMR：400MHz CDCl3δ7.43-7.41(m，6H)，7.29-7.20(m，7H)，4.57(d，J＝2.0Hz，2H)，3.45(s，2H)，3.31(s，2H)，3.13(d，J＝7.6Hz，1H)，2.52(t，J＝6.0Hz，2H)，2.09(t，J＝6.8Hz，2H)，1.69(s，2H)，1.40(s，9H)。LCMS：(M+23)+：704.2
向7D(1.50g，2.20mmol)的THF溶液(20mL)加入DCC(590mg，2.86mmol)和N-羟基琥珀酰亚胺(329mg，2.86mmol)。反应搅拌12小时。反应混合物过滤，减压浓缩得到残余物，用硅胶层析柱纯化得到7E是白色固体(0.87g)。
1H NMR：400MHz CDCl3δ8.16(t，J＝6.4Hz，1H)，8.00(s，1H)，7.87(d，J＝8.0Hz，1H)，7.42-7.40(m，6H)，7.28-7.17(m，9H)，6.02(br，s，1H)，5.68(br，s，1H)，4.56(d，J＝6.0Hz，2H)，3.44(d，J＝12Hz，3H)，3.27(d，J＝5.6Hz，2H)，2.91(s，4H)，2.56-2.47(m，2H)，2.13-2.07(m，2H)，1.69(s，2H)，1.40(s，9H)
实施例11
人胰岛素(18mg，3.1μmol)和7E(2mg，2.57μmol)溶于DMSO(300μL)，加入DIEA(1.56μL，9μmol)，搅拌1小时。用RP-HPLC纯化。冷冻干燥后的固体加入TFA(2mL)，搅拌15分钟，加入20％乙腈溶液(5mL)，用RP-HPLC纯化，冷冻干燥后得到
Figure PCTCN2016110876-appb-000115
LCMS：[M+1]+＝6130.2
实施例12
以下结构的艾塞那肽前药用上述多肽合成法合成，RP-HPLC纯化。
Figure PCTCN2016110876-appb-000116
MS：[M+1]+＝4559.1
这个艾塞那肽前药可以与包含DBCO反应基团的水凝胶反应，装载在水凝胶上。
实施例13
生理盐水中的半衰期测量
多肽GIVEQAA-NH2和GIVEQAAY，两个胰岛素A链N末端部分的模拟，用作多肽模型测量以酰胺键连接多肽N末端的各种可降解连接基团的半衰期。多肽模型用Fmoc化学合成，用TFA切割，用制备HPC纯化。
由可降解连接基团封端的多肽模型溶解以1mg/mL的浓度溶解于生理盐水，在37℃水浴中培养。样品在不同的时间点取样分析(例如8h，16h，24h，48h，72h，96h，120h)。如果一个可降解连接基团封端的多肽模型的半衰期显著延长或者缩短，取样分析的时间框架要相应改变。加入0.1％TFA溶液使pH下降到2抑制可降解连接基团断开。RP-HPLC用于检测断裂反应。断裂的速率通过测量可降解连接基团封端的和释放的多肽模型的峰的面积定量研究。安捷伦6110四极LC-MS用于证实反应过程中的质量变化。
使用岛津LC-2010A HT系统和150mm x 4.6mm Zorbax 300SB-C18柱进行RP-HPLC分析。流速是1ml/min。溶剂A包含0.1％TFA/5％CH3CN去离子水，溶剂B包含0.1％TFA 100％CH3CN。使用线性梯度(例如10-70％B，10分钟)。
测定各个可降解连接基团封端的多肽模型的切割速率。可降解连接基团封端的多肽模型和释放的多肽模型的浓度用他们各自的峰面积测定。可降解连接基团封端的多肽模型的一级分离速率常数通过测绘不同时间点的[起始可降解连接基团封端的多肽模型]/[剩余可降解连接基团封端的多肽模型]的对数来测定。可降解连接基团封端的多肽模型的断裂半衰期通过使用绘图得到的公式和斜率计算。
连接到多肽模型的各种可降解连接基团的断裂半衰期可以用上述步骤测定。这些试验产生的数据见表1和2。
表1
连接多肽模型X-GIVEQAAY N末端的各种可降解连接基团的断裂
Figure PCTCN2016110876-appb-000117
表2
连接多肽模型X-GIVEQAA N末端的各种可降解连接基团的断裂
Figure PCTCN2016110876-appb-000118
Figure PCTCN2016110876-appb-000119
下面的化合物的断裂半衰期是779小时。
Figure PCTCN2016110876-appb-000120
实施例14
血清中半衰期测量
分析中使用可降解连接基团封端的多肽模型X-G-dI-dV-dE-dQ-dA-dA。使用D型氨基酸以避免对多肽模型酶切。多肽溶解于100％血清，在37℃培养。样品在不同的时间点取样分析(例如8h，16h，24h，48h，72h，96h，120h)。如果一个可降解连接基团封端的多肽模型的半衰期显著延长或者缩短，取样分析的时间框架要相应改变。
测定各个可降解连接基团封端的多肽模型的切割速率。可降解连接基团封端的多肽模型和释放的多肽模型的浓度用他们各自的峰面积测定。可降解连接基团封端的多肽模型的一级分离速率常数通过测绘不同时间点的[起始可降解连接基团封端的多肽模型]/[剩余可降解连接基团封端的多肽模型]的对数来测定。可降解连接基团封端的多肽模型的断裂半衰期通过使用绘图得到的公式和斜率计算。
表3
连接多肽模型X-G-dI-dV-dE-dQ-dA-dA N末端的各种可降解连接基团的断裂
Figure PCTCN2016110876-appb-000121
Figure PCTCN2016110876-appb-000122
实施例15
Figure PCTCN2016110876-appb-000123
化合物1I(100mg，0.2mmole)，DEPBT(60mg，0.2mmole)，DIPEA(35μL，0.2mmole)逐次加入6C(44.5mg，0.15mmole)的DMSO(2mL)溶液，室温搅拌过夜。混合物用乙酸乙酯(100mL)和水(100mL)稀释。水层用乙酸乙酯萃取(100mL x 2)。合并的有机层用饱和食盐水(100mL)洗，用无水硫酸钠干燥，真空浓缩。残余物溶于50％哌啶/DMF(3mL)，室温搅拌15分钟。混合物用乙酸乙酯(20mL)和水(20mL)稀释。水层用乙酸乙酯萃取(15mL x 2)。合并的有机层用饱和食盐水(10mL)洗，用无水硫酸钠干燥，真空浓缩。残余物用硅胶柱纯化。洗脱液真空浓缩得到10A(61mg，91％产率)。10A(61mg)和DIEA(48mg，65μl)缓缓加入DBCO-PEG4-NHS(CAS：1427004-19-0，259.6mg，0.4mmole)的二氯甲烷(2mL)溶液，室温搅拌1小时。蒸发溶剂，加入50％TFA/二氯甲烷(2mL)，搅拌15分钟后蒸发溶剂。残余物用快速色谱法纯化，得到10B(183mg)。
实施例16
3-叠氮基丙酸(202.5mg，1.76mmole)、DEPBT(528mg，1.76mmole)和DIEA(306.6μL，1.76mmole)加入PEG5K-(NH2)8(1g，0.2mmole)的二氯甲烷溶液(15mL)，室温搅拌8小时。反应溶液浓缩到5mL，加入MTBE(30mL)搅拌15分钟，固体沉淀过滤，真空干燥，得到PEG5K-[NHC(O)-CH2CH2N3]8(1.03g)。
实施例17
10B(10mg)加入PEG5K-[NHC(O)-CH2CH2N3]8(10mg)的生理盐水溶液(400μL)，得到水凝胶17A。10B(20mg)加入PEG5K-[NHC(O)-CH2CH2N3]8(10mg)生理盐水溶液(400μL)，得 到水凝胶17B。水凝胶17A和17B分别悬浮于生理盐水，在37℃水浴中培养，按照固定的时间间隔取样测HPLC，使用Phenomenex Bio-Sep SEC-S2000 4.6X300mm 5μM凝胶过滤色谱柱。17A的降解时间为301小时，17B的降解时间是487小时，说明交联密度增加，水凝胶的降解速率下降。
实施例18
实施例7中得到的水凝胶(5mg)悬浮于PBS中，加入8A(6mg)，室温反应30分钟后，除去上清液，用PBS洗，得到包含胰岛素的水凝胶，真空干燥。向水凝胶加入50％TFA/CH2Cl2(200μL)，15分钟后除去。水凝胶用乙腈(3X1mL)和纯净水洗。
为测量水凝胶中胰岛素的装载量，取胰岛素水凝胶(1mg)，在pH10水浴，使水凝胶上的胰岛素完全从水凝胶释放。样品中的胰岛素含量通过HPLC中胰岛素峰的面积与已知精确定量的胰岛素标准品在同样系统下的面积对比计算，胰岛素实际含量0.3毫克。包含胰岛素的水凝胶(1mg)悬浮于PBS(1ml)，在37℃水浴。每48小时取样一次。胰岛素在生理盐水中的释放半衰期根据实施例13中的方法测算。胰岛素的释放半衰期是197小时。
实施例19
实施例6中得到的水凝胶(10mg)悬浮于PBS中(2ml)，加入9A(5mg)。1小时后除去上清液，用纯净水洗，得到装载艾塞那肽的水凝胶。艾塞那肽水凝胶的艾塞那肽含量、艾塞那肽释放半衰期测量和计算方法与实施例18中的方法一样。每毫克水凝胶包含0.2毫克艾塞那肽。艾塞那肽释放半衰期143小时。
实施例20
实施例8中得到的水凝胶(100mg)悬浮于二氯甲烷(500μL)，加入MAL-dPEG4-NHS(Quanta Biodesign，1.59mg)和DIEA(2μL)。30分钟后加入TFA(500μL)，20分钟后除去上清液，水凝胶用二氯甲烷(2X1mL)、乙腈(2X1mL)和生理盐水(2X1mL)洗，然后悬浮于生理盐水(500μL)，加入8A(36.8mg)，室温反应30分钟后，除去上清液，加入9A(45.6mg)的生理盐水溶液，室温反应30分钟后，除去上清液，用纯净水洗(3X1mL)，得到装载胰岛素和艾塞那肽的水凝胶。
实施例21
重组尿酸氧化酶冻干粉(1mg)、10B(20mg)和PEG5K-[NHC(O)-CH2CH2N3]8(10mg)溶于生理盐水(400μL)，混匀后静置5小时，HPLC分析显示上清液中没有游离尿酸氧化酶，说明尿酸氧化酶已经被水凝胶封装。向尿酸氧化酶水凝胶加入人血清(600μL)，在37℃ 水浴中培养，定时取样测HPLC，使用Phenomenex Bio-Sep SEC-S2000 4.6X300mm 5μM凝胶过滤色谱柱。尿酸氧化酶完全释放的时间为416小时。拉布立酶(重组尿酸氧化酶)是短效药物，给药方式是静脉滴注。尿酸氧化酶水凝胶可以制成由患者自己皮下注射的长效剂型。
传统的蛋白封装水凝胶通常需要末端带有甲基丙烯酸酯等功能团的交联剂，蛋白在氧化还原体系下(例如过硫酸胺/N，N，N′，N′-四甲基乙二胺)完成封装。这样的反应和溶剂可能影响蛋白的正常功能。本发明使用的叠氮和炔基、氨基和琥珀酸亚胺、巯基和马来酰亚胺等关联反应对避免了这个风险，而且封装的蛋白的释放速率可以通过改变可降解连接基团、骨架分子(本实施例是8臂PEG)的分子量和交联物的长度、交联密度等调节。一般情况下，骨架分子量越大，交联物长度越长，水凝胶的孔径就越大，封装的蛋白更容易透出。交联密度也是调节水凝胶孔径的有效方法。
临床上需要静脉注射的蛋白药物很多，例如伊米苷酶、RIXUBIS(重组凝血因子IX)等。NAGLAZYME(Galsulfase制剂)每次静脉滴注时间至少4小时，因此需要在医疗机构进行。这些药物都可以采用本实施例类似的方法开发出患者可以自己皮下注射的长效水凝胶制剂，改善治疗效果和使用体验。。
实施例22
Fmoc-Lys(Mtt)-OH(250mg，400μmole)，DEPBT(120mg，400μmole)，DIPEA(140μL，800μmole)加入PEG20K-(NH2)4(2g，100μmole)的二氯甲烷溶液(25mL)，反应在室温搅拌6小时后，加入醋酸酐(50μL，526μmole)，继续搅拌10分钟。反应溶液浓缩到10mL，加入MTBE(80mL)搅拌10分钟，过滤固体沉淀，真空干燥，直接用于下一步反应。
[Fmoc-Lys(Mtt)-NH]4-PEG20K(2.02g，90μmole)溶于DMF(15mL)，加入哌啶(1mL，10mmol)。反应在室温搅拌15分钟后，加入MTBE(80mL)，过滤后得到产物[H-Lys(Mtt)-NH]4-PEG20K(1.76g，91％)。
Figure PCTCN2016110876-appb-000124
1I(176mg，350μmole)，DEPBT(105mg，350μmole)和DIEA(61μL，360μmole)加入[H-Lys(Mtt)-NH]4-PEG20K(1.76g，82μmole)的DMF(15mL)溶液，室温搅拌6小时。减压除去溶剂后，加入50％哌啶/DMF(10mL)，搅拌15分钟。减压除去溶剂后，残余物用HPLC纯化得到22A(1.59g)。
5-叠氮基戊酸(50mg，350μmole)，DEPBT(105mg，350μmole)和DIEA(61μL，350μmole)加入22A(1.59g)的DMF(15mL)溶液，室温搅拌8小时。减压除去大部分溶剂后，加入MTBE(50mL)，搅拌10分钟，过滤固体沉淀，溶于1％TFA/CH2Cl2(10mL)，搅拌30分钟。溶液浓缩到5mL，加入MTBE(30mL)，搅拌10分钟，过滤固体沉淀，真空干燥，得到22B(1.24g)，直接用于下一步反应。
碘代醋酸(60mg，320μmole)，DEPBT(96mg，320μmole)和DIEA(56μL，320μmole)加入22B(1.14g)的DMF(12mL)溶液，室温搅拌过夜。加入MTBE(100mL)，搅拌10分钟，过滤固体沉淀，真空干燥，得到22C(1.03g)，直接用于下一步反应。
22C(1g)溶于CH2Cl2(4mL)，加入TFA(2mL)。反应在室温搅拌10分钟，加入MTBE(20mL)，沉淀过滤后用HPLC纯化，得到白色粉末22E(0.9g)。
实施例23
22E(100mg)加入实施例2的PEG40K(NHC(O)CH2l)4(DBCO)4(200mg)的生理盐水溶液(6mL)，混合均匀，静置6小时，去除上清液，连续用纯净水(10mL)、乙醇(10mL)和乙腈(10mL)洗，真空干燥，得到水凝胶23A。
22B(100mg)溶于50％TFA/CH2Cl2(1mL)，室温搅拌15分钟，减压除去溶剂。残余物溶于二氯甲烷(1mL)，加入MTBE(6mL)，搅拌10分钟，固体沉淀过滤，加入PEG40K(NHC(O)CH2l)4(DBCO)4(200mg)的生理盐水溶液(6mL)，混合均匀，静置6小时， 去除上清液，连续用纯净水(10mL)、乙醇(10mL)和乙腈(10mL)洗，真空干燥，得到水凝胶23B。
实施例24
实施例23所得水凝胶23A(20mg)悬浮于生理盐水中，加入8A(20mg)，反应过夜，除去上清液，用生理盐水洗(2X3mL)，得到包含胰岛素的水凝胶23A-胰岛素。水凝胶23B(20mg)悬浮于生理盐水中，加入8A(20mg)，反应过夜，除去上清液，用生理盐水洗(2X3mL)，得到包含胰岛素的水凝胶23B-胰岛素。
为测量水凝胶中胰岛素的装载量，23A-胰岛素和23B-胰岛素在pH 10水浴，使水凝胶上的胰岛素完全从水凝胶释放。样品中的胰岛素含量通过HPLC中胰岛素峰的面积与已知精确定量的胰岛素标准品在同样系统下的面积对比计算。23A释放胰岛素10.5mg，23B释放4.7mg。说明在水凝胶的交联物上增加反应功能团可以有效增加水凝胶的药物装载量。
实施例25
透明质酸(MW：100K，200mg)溶于0.1M MES缓冲液(100mL)，加入EDC(144mg，0.75mmole)和N-羟基琥珀酰亚胺(86mg，0.75mmole)，搅拌30分钟。N-(2-氨乙基)马来酰亚胺盐酸盐(110mg，0.63mmole)溶于0.1M MES缓冲液(40mL)，加入透明质酸反应。4小时后，反应混合物先后用20nM氯化钠溶液和去离子水透析，冷冻干燥得到白色泡沫状固体。透明质酸-马来酰亚胺的1H-NMR(D2O)图谱显示δ7.0峰，对应于马来酰亚胺的乙烯基质子。透明质酸-马来酰亚胺的取代度定义为每100个透明质酸二糖环的马来酰亚胺的数量，通过比较对应于透明质酸乙酰胺甲基质子和马来酰亚胺的乙烯基质子δ2.0和δ7.0来确定。以上反应测得取代度为8。减少反应中N-(2-氨乙基)马来酰亚胺与透明质酸的比例，可以降低取代度。
透明质酸-马来酰亚胺与PEG10K-(SH)4溶于生理盐水，1-3％w/v，巯基与马来酰亚胺的比例1.1。随着浓度增加，凝胶时间缩短。当取代度为4时，1％w/v凝胶时间为40s，3％w/v凝胶时间为15s。取代度增加，凝胶时间也缩短。当取代度为8时，3％w/v凝胶时间为7s。
透明质酸-马来酰亚胺、PEG10K-(SH)4和8A溶于生理盐水，马来酰亚胺：PEG巯基：8A巯基的比例1∶0.8∶0.3，1％w/v，混匀静置1小时，得到透明质酸-马来酰亚胺-8A，可用于皮下注射给药。
实施例26
9周龄雄性Wistar大鼠，平均体重240±10克，安置于聚丙烯笼中，保持恒温恒湿，12小时光照/黑暗周期，自由摄取食物和饮水。糖尿病诱导时，大鼠禁食48小时，腹腔内注射链脲霉素(60毫克/公斤，溶于10nM柠檬酸钠缓冲剂，pH 4.5)，然后提供食物。三天后检查血糖。血糖高于250毫克/分升定义为糖尿病，血糖高于450毫克/分升的大鼠用于实验。每天注射重组胰岛素(24纳摩尔/公斤)，以保持血糖不超过550毫克/分升。
胰岛素水凝胶按实施例8和实施例20方法制备，但用于本实施例的水凝胶只装载胰岛素。胰岛素实际含量按实施例18方法测算。
高血糖(400-450毫克/分升)大鼠分为3组，每组8只。第一组大鼠腹腔注射生理盐水(100μL)，第二组大鼠每天腹腔注射40纳摩尔/公斤地特人胰岛素。第三组大鼠注射水凝胶(75纳摩尔/公斤，按照水凝胶中人胰岛素实际含量计算)。另外一组8只健康大鼠每天腹腔注射生理盐水(100μL)作为对照。上午11点测量。结果如图1所示。
地特胰岛素是一种代表性的长效胰岛素，临床使用是一天一针。胰岛素水凝胶皮下注射一次，降糖效果至少保持一周。地特胰岛素由于使用脂肪酸长效修饰，生物活性降低，因此在40纳摩尔/公斤剂量无法有效降低血糖。胰岛素水凝胶释放的胰岛素保留了全部活性，以比地特胰岛素更低的剂量降低血糖接近正常值。另外，胰岛素水凝胶没有像很多其它的水凝胶一样，在初期显示“突释”现象，没有出现过多胰岛素导致的低血糖。胰岛素水凝胶组动物的血糖在1-7天基本平稳，说明胰岛素是以均匀的速度释放。本实施例说明本发明的水凝胶可以成为一种有效的长效给药技术。

Claims (23)

  1. 一种可在生理条件下降解的水凝胶，其特征在于：所述水凝胶包括至少一种骨架和交联物，所述交联物中包括可降解连接基团，所述可降解连接基团通过分子内环化反应降解，从而使水凝胶水降解成为水溶性的、较小分子量成份。
  2. 如权利要求1所述的水凝胶，其特征在于：所述可降解连接基团包括可以在生理条件下断裂的部分，还包括与一种或多种反应性聚合物形成共价键的反应基团；所述可以在生理条件下断裂的部分具有式
    Figure PCTCN2016110876-appb-100001
    其中X选自OH或-HN-R0；
    Y选自：
    (1)N-RO；
    (2)C(RpRq)；
    (3)O或S，条件是X不是OH；
    (4)C(RpRq)，当X是-HN-R0，其中R0和Rp连同与其相连的原子可以组成4、5或6元杂环；
    Y1选自
    (1)C(R3R4)；
    (2)C(O)或C(S)，条件是A不是C(O)，C(S)，SO，or SO2；
    (3)O，S，SO或SO2，条件是Y和A不是O；
    (4)-N-Rn；和
    (5)键；
    J1是-C(R10R11)或共价键；
    波浪线是可降解连接基团与生物活性物质或者反应性聚合物形成酰胺键或者酯键的连接点；
    A和B共同构成环系统(ring system)，包括：有6-15个碳原子的芳基；有3，4，5，6，7，8，9或10元环的环烷基；有4，5，6，7，8，9，或10元环的环烯基；有5，6，7，8，9或10元环的环炔基；饱和和不饱和单环、多环和稠环；饱和和不饱和单杂环、多杂环和稠杂环，每个杂环包含一个或多个杂环原子N，S或O，而且每个环是3元环到8元环；
    前述的每一个环可选地被一个或多个基团取代，基团选自低级烷基(lower alkyl)、低级烷氧基(lower alkoxy)、酰基、酰氧基、烷氧羰基，芳基、苯基、苄基、卤素、卤代甲酰基、卤代低级烷基(haloloweralkyl)、三氟甲基、三氟甲氧基、三氯甲基、氰酰、异 氰、异氰酸酯(或盐)、异硫氰酸酯、硫氰酸酯(或盐)、低级烷硫基(lower alkylthio)，氨基、亚氨基、氨基低级烷基(amino lower alkyl)、低级烷氨基(lower alkylamino)、低级烷氨基(lower dialkylamino)、羟基、羟烷基、硝基、腈、异腈、吡啶基、叠氮、羧基、羧酰氨基(carboxamido)、乙酸基、硫烷基(thiolalkyl)、碳酸酯(或盐)、氨基甲酸酯、低级烷氨基甲酰(loweralkylcarbamyl)、双低级烷氨基甲酰(diloweralkylcarbamyl)、磺酸基、磺胺、磺酸酯(或盐)、磺酰、亚砜、硫化物、二硫化物和巯基；
    或者A和B及其相连接的原子共同形成一个芳香环，可选地被至少一个以上定义的功能团取代；或者A和B及其相连接的原子共同组成一个多芳环，可选地被至少一个以上定义的功能团取代；
    Y、A、B中至少包含一个可以与反应性聚合物形成共价键的反应基团，以此连接至一种或多种反应性聚合物；
    R0、RO、R3、R4、R10、R11、Rn、Rp、Rq各独立选自氢原子、低级烷基、低级烷氧基、酰基、酰氧基、烷氧羰基、芳基、苯基、苄基、卤素、卤代甲酰基、卤代低级烷基、三氟甲基、三氟甲氧基、三氯甲基、氰酰、异氰、异氰酸酯(或盐)、异硫氰酸酯、硫氰酸酯(或盐)、低级烷硫基、氨基、亚氨基、氨基低级烷基、低级烷氨基、低级烷氨基、羟基、羟烷基、硝基、腈、异腈、吡啶基、叠氮、羧基、羧酰氨基、乙酸基、硫烷基、碳酸酯(或盐)、氨基甲酸酯、低级烷氨基甲酰、双低级烷氨基甲酰、磺酸基、磺胺、磺酸酯(或盐)、磺酰、亚砜、硫化物、二硫化物和巯基；
    或者R0，RO，R3，R4，R10，R11，Rn，Rp，Rq各独立选自-SO2-OH，-SO2-NRm1Rm2，-SO2-Rm3，-O-Rm4，-S-Rm5，-N-Rm6Rm7，-C(O)Rm8，-C(O)ORm9，-OC(O)Rm10，-NHC(O)Rm11，-C(O)NRm12Rm13，-NHC(O)NRm14Rm15，其中Rm1，Rm2，Rm3，Rm4，Rm5，Rm6，Rm7，Rm8，Rm9，Rm10，Rm11，Rm12，Rm13，Rm14和Rm15独立选自氢原子(H)、(C1-C18)烷基、芳基、(C1-C18烷基)OH、(C1-C18烷基)SH、(C1-C18烷基)COOH、(C1-C18烷基)NH2、(C0-C4烷基)(C5-C6环烷基)、(C0-C10烷基)(C5-C6杂环)、(C0-C4烷基)(C6-C10芳基)、(C0-C4烷基)(C4-C9杂芳基)；
    或者Rp和Rq各独立选自C1-C20烷基、(C1-C10烷基)OH、(C1-C10烷基)SH、(C2-C3烷基)SCH3、(C1-C4烷基)CONH2、(C1-C10烷基)COOH、(C1-C10烷基)NH2、(C1-C4烷基)NHC(NH2 +)NH2、(C0-C4烷基)(C3-C6环烷基)、(C0-C4烷基)(C2-C5杂环)、(C0-C4烷基)(C6-C10芳基)、(C0-C4烷基)(C6-C10芳基)R16、(C1-C4烷基)(C3-C9杂芳基)、C1-C12烷基(W1)C1-C12烷基，其中W1是选自N、S和O的一个杂原子，R16选自H、OH、卤素、C1-C7烷基、C2-C7烯基、C2-C7炔基、CO2H，CO2(C1-C7烷基)、NH(C0-C10烷基)、O(C1-C10烷基)、杂芳基。
  3. 如任一在先权利要求所述的水凝胶，其水凝胶骨架选自以下物质的均聚物或共聚物：聚乙二醇、聚丙二醇、聚(N-乙烯吡咯烷酮)、聚甲基丙烯酸酯、聚磷腈、聚交酯、聚丙烯酰胺、聚乙醇酸、聚乙烯亚胺、琼脂糖、葡聚糖、明胶、胶原、聚赖氨酸、壳聚糖、藻酸盐、透明质酸、果胶、角叉聚糖、聚氨基酸，其在天然状态下具有合适的反应功能团和交联功能团，或经衍生从而具有合适的反应功能团和交联功能团。
  4. 如任一在先权利要求所述的水凝胶，其水凝胶的骨架的通式是J(L1-B1)n1，其中J是带分支的核；L1是从中延伸出来的链；B1具有至少一个交联功能团和至少一个可选的反应功能团；反应功能团和交联功能团可以相同，也可以不同；同一个骨架上的功能团不能相互反应；n1是2-32的整数。
  5. 如任一在先权利要求所述的水凝胶，其水凝胶骨架的通式是C(L1-B1)4，其中C是核心的季碳，L1是从中延伸出来的链，B1具有至少一个交联功能团和至少一个可选反应功能团。
  6. 如任一在先权利要求所述的水凝胶，其水凝胶骨架，L1是PEG基的聚合链；每个L1独立选自式-(CH2)n4(OCH2CH2)nLn5或(CH2)n4(CH2CH2O)nLn5，其中n4是1-5的整数，n是1-1000的整数，Ln5是连接L1末端和B1的键或者化学功能团。
  7. 如任一在先权利要求所述的水凝胶，其水凝胶骨架的通式是(C1-L1)n2J(L1-C2)n3，其中J是带分支的核；L1是从中延伸出来的链；C1是反应功能团，C2是交联功能团；反应功能团和交联功能团可以相同，也可以不同；同一个骨架上的功能团不能相互反应；n2和n3是0-32的整数。
  8. 如任一在先权利要求所述的水凝胶，其水凝胶，骨架部分的结构是(C1-L1)4J(L1-C2)4，其中J是带分支的核，L1是从中延伸出来的链，C1是反应功能团，C2是交联功能团。
  9. 如任一在先权利要求所述的水凝胶，其水凝胶骨架，L是PEG基的聚合链；每个L独立选自式-(CH2)n4(OCH2CH2)nLn5或(CH2)n4(CH2CH2O)nLn5，其中n4是1-5的整数，n是1-1000的整数，Ln5是连接L末端和C1或C2的键或者化学功能团。
  10. 如任一在先权利要求所述的水凝胶，交联物通式是B2-Z2-L2-Z2-B2，或者B2-Z2-L2-Z2-B2′，其中L2是连接两端的链，B2和B2′分别具有至少一个交联功能团和至少一个可选的反应功能团；Z2是可选的可生物降解连接基团，每一端的Z2可以相同，也可以不同；同一个交联物上的功能团不能相互反应。
  11. 如任一在先权利要求所述的水凝胶，其交联物，B2或B2′的分支结构中可以包含一个或一个以上可选的可生物降解连接基团。
  12. 如任一在先权利要求所述的水凝胶，交联物通式是B2-L2-B2，或者B2-L2-B2′，其中L2是连接两端的链，B2和B2′具有至少一个交联功能团和至少一个可选的反应功能团，交联功能团和反应功能团分别可以与可选的可降解连接基团相连；同一个交联物上的功能团不能相互反应。
  13. 如任一在先权利要求所述的水凝胶，交联物的通式是O(L2-Z2-B2)n6，其中O是带分支的核，L2是从中延伸出来的链，n6是2-16的整数，Z2是可选的可降解连接基团；B2具有至少一个交联功能团和至少一个可选的反应功能团，反应功能团可以通过可选的可降解连接基团相连到B2。
  14. 如任一在先权利要求所述的水凝胶，其交联物，L2是PEG基的聚合链，两端分别通过共价键连接带分支的核和B2；每个L2独立选自式-(CH2)n4(OCH2CH2)nLn5，其中n4是1-5的整数，n是1-1000的整数，Ln5是连接L2末端和Z2的键或者化学功能团。
  15. 如任一在先权利要求所述的水凝胶，其交联物，B2的分支结构中可以包含一个或一个以上可选的可生物降解连接基团。
  16. 如任一在先权利要求所述的水凝胶，交联物的通式是O(L2-B2)n6，B2具有至少一个交联功能团和至少一个可选的反应功能团，互联功能团和反应功能团可以分别与可选的可降解连接基团相连。
  17. 如任一在先权利要求所述的水凝胶，所述可以在生理条件下断裂的部分包含的6元环的化学式如下：
    Figure PCTCN2016110876-appb-100002
    其中U1，U2，U3，U4，U5和U6独立选自CR12和N；
    其中X选自-OH或-HN-R0；
    Y选自：
    (1)N-RO；
    (2)C(RpRq)；
    (3)O，条件是X不是OH；
    (4)C(RpRq)，当X是HN-R0，其中R0和Rp连同与其相连的原子可以组成4，5或6元杂环；
    Y1选自
    (1)C(R3R4)；
    (2)C(O)or-C(S)，条件是Y和A不是C(O)，C(S)，SO或SO2；
    (3)O，S，SO或SO2，条件是Y和A不是O；
    (4)N-Rn；和
    (5)键；
    J1是-C(R10R11)或共价键；
    R12选自氢原子、低级烷基、低级烷氧基、酰基、酰氧基、烷氧羰基、芳基、苯基、苄基、卤素、卤代甲酰基、卤代低级烷基、三氟甲基、三氟甲氧基、三氯甲基、氰酰、异氰、异氰酸酯(或盐)、异硫氰酸酯、硫氰酸酯(或盐)、低级烷硫基、氨基、亚氨基、氨基低级烷基、低级烷氨基、低级烷氨基、羟基、羟烷基、硝基、腈、异腈、吡啶基、叠氮、羧基、羧酰氨基、乙酸基、硫烷基、碳酸酯(或盐)、氨基甲酸酯、低级烷氨基甲酰、双低级烷氨基甲酰、磺酸基、磺胺、磺酸酯(或盐)、磺酰、亚砜、硫化物、二硫化物和巯基；
    或者R12选自-SO2-OH，-SO2-NRm1Rm2，-SO2-Rm3，-O-Rm4，-S-Rm5，-N-Rm6Rm7，-C(O)Rm8，-C(O)ORm9，-OC(O)Rm10，-NHC(O)Rm11，-C(O)NRm12Rm13，-NHC(O)NRm14Rm15，其中Rm1，Rm2，Rm3，Rm4，Rm5，Rm6，Rm7，Rm8，Rm9，Rm10，Rm11，Rm12，Rm13，Rm14和Rm15独立选自氢原子(H)、(C1-C18)烷基、芳基、(C1-C18烷基)OH、(C1-C18烷基)SH、(C1-C18烷基)COOH、(C1-C18烷基)NH2、(C0-C4烷基)(C5-C6环烷基)、(C0-C10烷基)(C5-C6杂环)、(C0-C4烷基)(C6-C10芳基)、(C0-C4烷基)(C4-C9杂芳基)。
  18. 如任一在先权利要求所述的水凝胶，Y1和J1分别是共价键，所述可以在生理条件下断裂的部分包含的6元环的化学式如下：
    Figure PCTCN2016110876-appb-100003
    其中X选自-OH或-HN-R0；
    Y选自：
    (1)N-RO；
    (2)C(RpRq)；
    (3)O，条件是X不是OH；
    (4)C(RpRq)，当X是-HN-R0，其中R0和Rp连同与其相连的原子可以组成4，5或6元杂环；
    U1，U2，U3，U4，U5，U6，R0，RO，Rp和Rq的定义如上。
  19. 如任一在先权利要求所述的水凝胶，特征在于水凝胶包含生物活性物质，可以使用非共价储存库或共价储存库方法。
  20. 如任一在先权利要求所述的水凝胶，特征在于生物活性物质与可降解连接基团相连，而可降解连接基团连接到水凝胶(共价储存库方法)。
  21. 如任一在先权利要求所述的水凝胶，生物活性物质与可降解连接基团的结构是
    Figure PCTCN2016110876-appb-100004
    ，通过巯基与水凝胶相连。
  22. 如任一在先权利要求所述的水凝胶，生物活性物质与可降解连接基团的结构是
    Figure PCTCN2016110876-appb-100005
    ，通过叠氮基团与水凝胶反应后相连。
  23. 一种制备释药型水凝胶的方法，所述方法包括以下步骤：
    (1)骨架的反应功能团先与反应功能团-可选可降解连接基团-生物活性物质反应，得到中间体载药聚合物；
    (2)任选地纯化中间体载药聚合物；
    (3)骨架的交联功能团与交联物反应生成释药可降解水凝胶。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112638434A (zh) * 2018-06-28 2021-04-09 哈鲁拉公司 交联的多羧酸化多糖及其使用方法
US20210139863A1 (en) * 2019-11-11 2021-05-13 Gwangju Institute Of Science And Technology Conjugates of fatty acid-therapeutic proteins for half-life extension and use of the same

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109796608A (zh) * 2019-01-21 2019-05-24 福建农林大学 一种pH响应的具粘附性、抗菌、耐压的快速自修复胶原基水凝胶的制备方法
CN112111162B (zh) * 2019-06-21 2022-03-08 中国科学院苏州纳米技术与纳米仿生研究所 可快速固化的双网络水凝胶及其制备方法与应用
JP2024506853A (ja) * 2021-02-01 2024-02-15 ザ リージェンツ オブ ザ ユニバーシティ オブ コロラド，ア ボディー コーポレイト ハイブリッドネットワークヒドロゲルの作製および使用のための組成物および方法
CN113582770A (zh) * 2021-08-12 2021-11-02 吉林隆源农业服务有限公司 一种玉米种植用缓释长效复合肥及其制备方法
CN114409890A (zh) * 2022-02-28 2022-04-29 中国科学院长春应用化学研究所 一种氨基功能化的聚乙二醇衍生物及其制备方法
CN114767655B (zh) * 2022-04-19 2023-09-22 中国药科大学 一种两性离子功能化的生物可降解口服纳米载药系统及应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010059883A1 (en) * 2008-11-19 2010-05-27 Rutgers, The State University Of New Jersey Degradable hydrogel compositions and methods
CN102573913A (zh) * 2009-07-31 2012-07-11 阿森迪斯药物股份有限公司 可生物降解的基于聚乙二醇的水不溶性水凝胶
CN103945870A (zh) * 2011-09-07 2014-07-23 普罗林科斯有限责任公司 生物可降解交联的水凝胶

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3313765A (en) * 1963-05-10 1967-04-11 Celanese Corp Polymers containing oxymethylene and thiomethylene units
DE1225818B (de) * 1964-03-18 1966-09-29 Monsanto Co Verwendung wasserloeslicher Polyphosphonsaeure-verbindungen als Potenzierungsmittel fuer Bakterizide
US10626156B2 (en) 2013-12-06 2020-04-21 Jie Han Bioreversable promoieties for nitrogen-containing and hydroxyl-containing drugs

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010059883A1 (en) * 2008-11-19 2010-05-27 Rutgers, The State University Of New Jersey Degradable hydrogel compositions and methods
CN102573913A (zh) * 2009-07-31 2012-07-11 阿森迪斯药物股份有限公司 可生物降解的基于聚乙二醇的水不溶性水凝胶
CN103945870A (zh) * 2011-09-07 2014-07-23 普罗林科斯有限责任公司 生物可降解交联的水凝胶

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PENG HUI ET AL.: "Synthesis and characterization of novel biodegradable amphoteric ph-sensitive hydrogel", MATERIAL REVIEW, vol. 24, no. 7, 31 July 2010 (2010-07-31), pages 48 - 51, ISSN: 1005-023x *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112638434A (zh) * 2018-06-28 2021-04-09 哈鲁拉公司 交联的多羧酸化多糖及其使用方法
CN112638434B (zh) * 2018-06-28 2023-08-15 哈鲁拉公司 交联的多羧酸化多糖及其使用方法
US20210139863A1 (en) * 2019-11-11 2021-05-13 Gwangju Institute Of Science And Technology Conjugates of fatty acid-therapeutic proteins for half-life extension and use of the same

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