HK1176311B - Sterilization of biodegradable hydrogels - Google Patents

Description

Disinfection of biodegradable hydrogels

Biodegradable PEG-based hydrogels are of interest for various medical and pharmaceutical applications such as tissue regeneration, wound closure and drug delivery. For safety reasons, it is highly preferred to incorporate engineered biodegradability in PEG hydrogels in certain applications, such as drug delivery. Biodegradability can be introduced into hydrogels by ester bonds that undergo spontaneous or enzymatic hydrolysis in aqueous or in vivo environments.

As a corresponding commercial product, the sterility of pharmaceutical compositions or medical devices for implantation or topical application must be certified. Various sterilization methods have been proposed, such as heat, pressure, filtration, chemical treatment or irradiation. Unfortunately, these sterilization methods cannot be used with biodegradable PEG hydrogels because they are incompatible with the maintenance of hydrogel structure and properties, thereby limiting the medical applications of biodegradable PEG hydrogels.

For example, injectable solutions are most commonly sterilized in their vials by autoclaving, but the biodegradable bonds are rapidly degraded if performed at elevated temperatures. Thus, autoclaving a biodegradable PEG hydrogel would result in a pre-degraded material that does not compound the requirements of medical applications.

Alternatively, the solution may be sterilized by filtration using a filter having a pore size of 0.2 μm to remove any microbial contaminants, and then the sterile solution is filled into a vial under sterile conditions. However, in the case of insoluble cross-linked PEG hydrogels, the material is insoluble, but may exist in the form of a suspension of particles, typically greater than 0.2 μm in size, or as other three-dimensional objects (such as discs or tubes), and therefore such suspensions or gels cannot be sterilized by filtration.

International patent application WO2003/035244 describes a closed circuit apparatus capable of producing sterile microparticles. Throughout the particle formation process, the chemical components sterilized by the filter are maintained sterile in a sterile environment, thereby resulting in sterile microparticles.

This aseptic process, which uses a sterile filtration step at the raw material level and maintains aseptic conditions during the process, has certain disadvantages compared to a process in which sterilization is performed after hydrogel synthesis (referred to as terminal sterilization). The earlier the sterilization step occurs in the production process, the higher the risk of accidental contamination. Aseptic processing also requires precision industrial equipment, thereby increasing production costs. Therefore, terminal sterilization methods are preferred.

Photodegradation of polymers by UV light or gamma irradiation can generate free radicals and/or ions that often lead to fragmentation and crosslinking. Since the case where light exposure is performed in the absence of oxygen rarely occurs, oxidation also occurs, which complicates the case. This generally changes the properties of the hydrogel and the susceptibility of the material to biodegradation (Encyclopedia of Polymer Science and Technology, Mark Herman (eds.) Wiley, 2004, p 263 and subsequent pages).

Treatment with ethylene oxide gas or with a solution containing hydrogen peroxide can lead to similar side reactions which can adversely affect the biodegradation properties of the hydrogel and lead to a significant deviation in the degradation kinetics of the treated hydrogel compared to the untreated hydrogel material. Furthermore, care must be taken to ensure that no significant amounts of, for example, ethylene oxide remain in the hydrogel, which can be toxic and lead to undesirable side effects.

To avoid the difficulties associated with terminal sterilization, cross-linking and sterilization processes have been combined. US5,634,943 details a method for producing cross-linked PEG hydrogels by gamma irradiation. In this method, linear PEG (MW 200kDa) is dissolved in saline, degassed and irradiated by a gamma source such as Co 60. The dose of 2.5-25 mrad (equivalent to 25-250KGy) is sufficient to crosslink the PEG chains by forming free radicals and interchain linkages, thereby obtaining a hydrated insoluble hydrogel. Due to the fact that the irradiation dose is also high enough to sterilize, a material suitable for implantation in the cornea of an eye is obtained in one step.

Similarly, U.S. patent application US20090030102 describes a method for forming crosslinked polymer gels for use in electronic components based on polyalkylene oxides, polyarylene oxides or polyglyceryl ethers, in which crosslinking is carried out by UV and/or gamma irradiation in the presence of a crosslinking agent and an organic solvent.

For other applications, such as drug delivery, biodegradability of PEG hydrogels is desirable. US6,537,569 details a method for producing degradable PEG hydrogels by gamma irradiation. Here, linear PEG chains (MW 10kDa) linked by biodegradable ester bonds are used. Irradiation with 25 or 30kGy formed interchain crosslinked structures and insoluble PEG hydrogels.

Attempts have also been made to control the kinetics of drug release by varying the degree of cross-linking in PEG hydrogels irradiated with UV or gamma Radiation (Minkova et al, J.Polym.Sci., Polym.Phys.27(1989) 621-642; Belcheva et al, Macromol. Symp.103(1996) 193; Rosiak and Yoshii, nucleic instruments and Methods in Physics research B151 (1999) 56-64; Rosiak and Ulanky, Radiation Physics and Chemistry 55(1999) 139-151; Dimitrov et al, Acta pharmaceutical Turcica 46(2004) 49-54). However, in this case the presence of the drug is required during the irradiation process to provide entrapment of the drug, but there is a possibility that irradiation induced side reactions such as oxidation or hydrolysis or binding to the polymer chains occur, which makes this approach impractical for most drugs.

It has also been shown that irradiation affects other properties of PEG-based hydrogels such as swelling and roughness (Kanjickal et al, J Biomed Mater Res a., 2008, 1/9 days — Effects of polymerization on poly (ethylene glycol) hydrogels).

Various PEG-based hydrogels have been described in the literature. For example, WO2006/003014 describes a polymer hydrogel conjugate of a prodrug, wherein the hydrogel is composed of non-biodegradable backbone moieties interconnected by cross-linkers comprising biodegradable bonds.

European patent application EP09167026.5 describes a PEG-based hydrogel with characteristic late-explosive degradation kinetics.

EP1019446 describes a hydrogel which consists only of PEG moieties. Hydrolytically unstable bonds are incorporated into the hydrogel and are thus capable of degradation. The patent also claims the use of such hydrogels as drug delivery systems.

Us patent 5,514,379 describes, inter alia, PEG-based hydrogels that may contain diagnostic labels, either alone or together with therapeutic drugs. Similarly, U.S. patent 6,602,952 describes PEG-chitosan hydrogels containing bioactive agents that can be injected into the body. PCT application WO2006/38462 describes hydrogels containing polyethylene oxide and having a urethane cross-linked structure for use as drug delivery devices or with other biomedical functions.

Although all of the above hydrogels are intended for applications requiring sterility, these patents or patent applications do not address this problem, which limits their industrial applicability.

The consequence of the disadvantage of hydrogel disinfection is that the insoluble crosslinked biodegradable PEG-based hydrogels are not recognized as such, but only as precursor compositions for the in situ formation of hydrogels after administration (Corgel)^TMThe hydrogel for the Bio is formed by a hydrogel,technology). Therefore, there is a need to provide a cost effective and conservative way to terminally sterilize hydrogels to adequately utilize the hydrogels in contamination sensitive applications.

It is therefore an object of the present invention to provide an alternative method for disinfection of insoluble biodegradable PEG-based hydrogels to at least partially overcome the above disadvantages and meet the above needs.

This object is achieved by a method for disinfecting a biodegradable polyethylene glycol-based insoluble hydrogel comprising backbone moieties interconnected by hydrolytically degradable bonds, said method comprising the steps of:

(a) providing a hydrogel;

(b) solvating the hydrogel in a protective solvent or in a mixture of two or more protective solvents or an aqueous solution thereof;

(c) the solvated hydrogel is gamma irradiated.

It has now surprisingly been found that pre-formed biodegradable PEG-based insoluble hydrogels can be sterilized by gamma irradiation and do not break unstable biodegradable bonds and thus also stable bonds when gamma irradiated in the presence of a protective solvent, preferably N-methyl-2-pyrrolidone (NMP), DMA, DMF or DMI, even more preferably NMP.

In particular, the in vitro degradation kinetics of such irradiated insoluble biodegradable PEG hydrogels of the present invention are the same as the in vitro degradation kinetics of non-irradiated PEG hydrogels. In addition, such irradiated insoluble biodegradable PEG hydrogels are still sufficiently degradable. If interchain crosslinks are created by the formation of free radicals, the degradation kinetics may be affected, and the degradation process of the insoluble biodegradable PEG hydrogel may be slower, the degradation curve flattens and degradation may be incomplete.

In the case where the insoluble biodegradable PEG-based hydrogel comprises functional groups, such groups remain functional after sterilization.

The terms used in the present invention have the following meanings.

Hydrogels can be defined as three-dimensional hydrophilic or amphiphilic polymer networks that can absorb large amounts of water. The network is composed of homopolymers or copolymers and is insoluble due to the presence of covalent chemical or physical cross-links (ionic, hydrophobic interactions, entanglement). The crosslinking provides network structure and physical integrity.

In the present context, the term "PEG-based hydrogel" ("PEG hydrogel") is understood to mean that the proportion by mass of PEG in the hydrogel is at least 10% by weight, preferably at least 25% by weight, based on the total weight of the hydrogel. The remainder may be made up of other polymers and other structural moieties.

The term "polymer" describes a molecule comprised of repeating structural units that are linked by chemical bonds in a linear, cyclic, branched, crosslinked, or dendritic fashion, or a combination thereof, which may be synthetic or of biological origin, or a combination of both. Examples include, but are not limited to, polyacrylic acid, polyacrylate, polyacrylamide, polyalkoxy polymer, polyamide, polyamidoamine, polyamino acid, polyanhydride, polyasparagine, polybutyranic acid, polycaprolactone, polycarbonate, polycyanoacrylate, polydimethylacrylamide, polyester, polyethylene glycol, polyethylene oxide, polyethyleneOxazoline, polyglycolic acid, polyhydroxyethyl acrylate, polyhydroxyethylOxazoline, polyhydroxypropylmethacrylamide, polyhydroxypropylmethacrylate, polyhydroxypropylacrylateOxazoline, polyiminocarbonate, poly (N-isopropylacrylamide), polylactic acid, poly (lactic-co-glycolic acid), polymethacrylamide, polymethacrylate, polymethyl methylAzolines, polypropylene fumarates, polyorganophosphazenes, polyorthoestersOxazoline, polypropylene glycol, polysiloxane, polyurethane, polyvinyl alcohol, polyvinyl amine, polyvinyl methyl ether, polyvinyl pyrrolidone, silicone, ribonucleic acid, deoxyribonucleic acid (desoxynecolic acid), albumins, antibodies and fragments thereof, plasma proteins, collagen, elastin, fascin (fascin), fibrin, keratin, polyaspartate, polyglutamate, prolamin, transferrin, cytochromes, flavoproteins, glycoproteins, hemoproteins, lipoproteins, metalloproteins, photosensitizers, phosphoproteins, opsin, agar, agarose, alginate, arabinose gum, arabinogalactan, carrageenan, cellulose, carboxymethylcellulose, hydroxypropylmethylcellulose and other carbohydrate-based polymers, chitosan, dextran, dextrin, gelatin, hyaluronic acid and derivatives, Mannan, pectin, rhamnogalacturonan, starch, hydroxyalkyl starch, xylan and copolymers and functionalized derivatives thereof.

"intact" in connection with the sterilized hydrogel means that no labile biodegradable bonds, and thus also no stable bonds, are destroyed during the sterilization process and no detectable further crosslinking takes place. The "intact" of the hydrogel can be determined by the in vitro degradation kinetics of the sterilized biodegradable PEG-based hydrogel according to the invention and at the same level as the in vitro degradation kinetics of the non-sterilized biodegradable PEG hydrogel. In addition, such irradiated biodegradable PEG hydrogels are still sufficiently degradable. If interchain crosslinks form by the formation of free radicals, the degradation kinetics may be affected, and the degradation process of the PEG hydrogel is slower, the degradation curve flattens, and degradation may be incomplete. The degradation process of PEG hydrogels is accelerated if cis chains are present. Preferably, the term "identical" in relation to two degradation kinetics means that the time required for said two degradation kinetics to obtain X% degradation differs by no more than 20%, preferably by no more than 15%, wherein X is between 5 and 90%.

If the biodegradable PEG-based hydrogel contains functional groups, these functional groups are retained. For example, if the functional group is an amine group, the amine content of the PEG-based hydrogel before the sterilization process is the same as after the sterilization process. Preferably, in the context of the present invention, the term "identical" means that the number of functional groups in the hydrogel sterilised according to the invention differs from the number of functional groups in the hydrogel before sterilisation by less than 30%, preferably by less than 20%.

To measure degradation kinetics, aliquots of soluble backbone degradation products can be separated from insoluble biodegradable PEG-based hydrogels and quantitatively measured in the absence of interference from other soluble degradation products released by the hydrogel. The hydrogel body may be separated from the excess water of the buffer at physiological osmolality by sedimentation or centrifugation. Centrifugation may be performed in such a way that the supernatant provides at least 10% by volume of swollen hydrogel. After this settling or centrifugation step, the soluble hydrogel degradation products may remain in the aqueous supernatant, and the water-soluble degradation products comprising one or more backbone moieties may be detected by subjecting an aliquot of this supernatant to suitable separation and/or analysis methods.

Alternatively, the water soluble degradation products can be separated from the water insoluble degradation products by filtration through a 0.45 μm filter and then the water soluble degradation products can be found in the flow through. The water soluble degradation products may also be separated from the water insoluble degradation products by a combination of centrifugation and filtration steps.

For example, the backbone moiety may carry a group that exhibits UV absorbance at wavelengths at which other degradation products do not exhibit UV absorbance. Such selective UV absorbing groups may be structural components of the backbone moiety, such as amide linkages, or may be incorporated into the backbone by attachment to its reactive functional groups via an aromatic ring system, such as an indolyl group.

In order to enhance the physicochemical or pharmacokinetic properties of drugs in vivo, these drugs may be combined with a carrier such as a hydrogel. If the drug is temporarily attached to the carrier and/or linker, such a system is often referred to as a carrier-linked prodrug. According to IUPAC (e.g. IUPAC)http://www.chem.qmul.ac.uk/ iupac.medchemGiven, 2009, day 7/22 visit), a carrier-linked prodrug is a prodrug containing a temporary bond of a given active substance to a temporary carrier group that can achieve enhanced physicochemical or pharmacokinetic properties and can be easily removed in vivo (typically by hydrolytic cleavage).

The terms "drug", "bioactive molecule", "bioactive moiety", "bioactive agent", "active agent" and the like are intended to mean any substance that can affect any physical or biochemical property of an organism, including but not limited to viruses, bacteria, fungi, plants, animals and humans. Bioactive molecules, as used herein, include, inter alia, any substance that is used to diagnose, treat, alleviate, treat or prevent a disease in a human or other animal, or to improve the physical or mental well-being of a human or animal. Examples of bioactive molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs (e.g., non-peptide drugs), dyes, lipids, nucleosides, oligonucleosides, polynucleotides, nucleic acids, cells, viruses, liposomes, microparticles, and micelles. Classes of biologically active agents suitable for use in the present invention include, but are not limited to, hypnotics and sedatives, psychostimulants, tranquilizers, respiratory drugs, anticonvulsants, muscle relaxants, anti-parkinson's (dopamine antagonists), analgesics, anti-inflammatories, anti-allergies, anxiolytics (anxiolytics), appetite suppressants, antiobesity agents, anti-migraine agents, muscle contractants, anti-infectives (antibiotics, antivirals, fungicides, antibacterials, vaccines), anti-inflammatories, antiarthritics, antimalarials, antiemetics, anti-epileptics, antidiabetics, bronchodilators, cytokines, growth factors, anticancer agents, anticoagulants, antihypertensives, cardiovascular agents, vasodilators, vasoconstrictors, antiarrhythmics, antiasthmatics, pharmaceutical packs, central nervous system active agents, hormones including contraceptives, anticoagulants, antipruritics, and anti-anxiety agents, Immunomodulators, sympathomimetics, diuretics, lipid regulating agents, antiandrogens, antiparasitics, anticoagulants, oncologics, antineoplastics, hypoglycemic agents, steroids, nutritional agents and supplements, growth supplements, anti-inflammatory agents, vaccines, antibodies, diagnostic agents, contrast agents, and the like.

By "small molecule bioactive moiety" is meant any of the above bioactive moieties having a molecular weight of 3000 daltons or less.

The biodegradability of the hydrogels used in the process of the present invention is achieved by the introduction of hydrolytically degradable bonds.

In the context of the present invention, the term "biodegradable" refers to a bond that is non-enzymatically hydrolytically degradable within a half-life of 1 hour to 3 months under physiological conditions (aqueous buffer at pH7.4, 37 ℃), and includes, but is not limited to, aconityl, acetal, carboxylic anhydride, carboxylic ester, imine, hydrazone, maleamic acid amide, orthoester, phosphoramide, phosphate, phosphosilicate, silyl ester, sulfonate, aromatic carbamate, combinations thereof, and the like. Preferred biodegradable linkages are carboxylate, carbonate, phosphate and sulfonate esters, most preferably carboxylate or carbonate. It will be appreciated that in practice, accelerated conditions, such as aqueous buffer at pH9, 37 ℃, may be used for in vitro studies.

Thus, hydrolyzable degradable bonds are, for example, aconityl, acetal, carboxylic anhydride, carboxylic ester, imine, hydrazone, maleamic acid amide, orthoester, phosphoramide, phosphate, phosphosilyl ester, silyl ester, sulfonate, aromatic carbamate, combinations thereof, and the like. Preferred hydrolytically degradable bonds are carboxylates, carbonates, phosphonates and sulfonates, most preferably carboxylates or carbonates.

The term "spacer" means that a urea, amide or carbamate group or ether is inserted between two carbons of a carbon chain.

"non-biodegradable" (stable) refers to a linkage that is a permanent bond that is not cleavable, meaning that the corresponding linking moiety has a half-life of at least 6 months under physiological conditions (aqueous buffer at pH7.4, 37 ℃).

By "sustained release delivery system" is meant a composition that releases a drug into a patient over an extended period of time.

By "surgical sealant" or "medical sealant" is meant a hydrogel-based adhesive and other devices used to close wounds such as incisions, lacerations, perforations, abrasions, contusions, or avulsions.

"hemostatic" refers to an agent used to stop bleeding from a wound.

By "surgical sponge" is meant a sponge used to absorb fluid from a surgical site.

Gamma radiation is defined as electromagnetic radiation having a quantum energy of more than 200keV, independent of its radiation source. The preferred radiation source is cobalt 60.

"sterile" means the absence of any detectable infectious agent, including bacteria, yeast, fungi, viruses, spores, in all stages and forms of development.

"Sterilization method" refers to a procedure that imparts sterility to a material, such as by irradiation, e.g., with UV or gamma radiation. Preferably gamma irradiation is used.

"protective solvent" describes a chemical compound used to solvate the dried hydrogel prior to sterilization to maintain the three-dimensional structure and physicochemical properties, and thus, to keep the hydrogel intact.

The present invention is explained in more detail below.

The present invention relates to a method for disinfecting biodegradable PEG-based insoluble hydrogels by irradiation in the presence of a protective solvent that keeps the hydrogel intact. The biodegradable PEG-based insoluble hydrogels sterilised according to the present invention have the same degradation kinetics and are fully degradable, which means that the labile biodegradable bonds are not destroyed and therefore also the stable bonds are not destroyed and undesired cross-linking does not occur, thereby leaving the hydrogel intact. If the biodegradable PEG-based insoluble hydrogel sterilized according to the present invention contains reactive functional groups, the functionality of these groups is also preserved, i.e., the groups remain intact. Such reactive functional groups can serve as a point of attachment for direct or indirect bonds of affinity ligands, chelating groups, drugs, prodrugs with attached carriers, and the like. Non-limiting examples of such reactive functional groups include, but are not limited to, carboxylic acids and activated derivatives, amino, maleimide, mercapto, sulfonic acids and derivatives, carbonates and derivatives, carbamates and derivatives, hydroxyl, aldehydes, ketones, hydrazines, isocyanates, isothiocyanates, phosphoric acids and derivatives, phosphonic acids and derivatives, haloacetyl, alkyl halides, acryloyl and other α, β -unsaturated michael acceptors, arylating agents such as aryl fluorides, hydroxylamines, disulfides such as pyridine disulfide, vinyl sulfones, vinyl ketones, diazoalkanes, diazoacetyl compounds, epoxides, ethylene oxide, and aziridines; preferred are carboxylic acids and activated derivatives, amino groups, mercapto groups, sulfonic acids and derivatives, carbonates and derivatives, carbamates and derivatives, hydroxy groups, aldehydes, ketones, hydrazines, isocyanates, isothiocyanates, phosphoric acids and derivatives, phosphonic acids and derivatives, haloacetyl groups, alkyl halides, acryloyl groups, arylating agents such as aryl fluorides, hydroxylamine, disulfide groups such as pyridine disulfide, vinyl sulfone, vinyl ketone, ethylene oxide and aziridine. Preferred reactive functional groups include mercapto, maleimide, amino, carboxylic acids and derivatives, carbonate and derivatives, carbamate and derivatives, aldehydes, and haloacetyl; more preferred are mercapto, amino, carboxylic acid and derivatives, carbonate and derivatives, carbamate and derivatives, aldehyde and haloacetyl. Preferably the reactive functional group is a primary amino group or a carboxylic acid, more preferably a primary amino group.

In one embodiment, the reactive functional groups of the biodegradable PEG-based insoluble hydrogel are protected by a cleavable protecting group upon sterilization.

PEG-based insoluble hydrogels sterilized according to the present invention may be used in any application where sterility is beneficial or desirable, such as tissue engineering, skin augmentation, intraocular devices, medical implants, surgical sealants and sponges, hemostats, sustained release drug delivery systems, medical imaging agents, and prodrug carriers. Preferred uses are sustained release drug delivery systems and prodrug-carriers, most preferably prodrug-carriers. The preformed three-dimensional hydrogel is sterilized by irradiation in a protective solvent or a mixture of two or more protective solvents or an aqueous solution thereof, and the sterile hydrogel may then optionally be loaded with, for example, a bioactive moiety such as a peptide, protein or small molecule. Such bioactive moieties may be linked to the hydrogel via a stable spacer moiety or by a degradable linker moiety.

In another embodiment, the biodegradable PEG-based insoluble hydrogel is first loaded with small molecule bioactive moieties and then sterilized by irradiation in the presence of a protective solvent or in a mixture of two or more protective solvents or aqueous solutions thereof.

PEG-based insoluble hydrogels suitable for sterilization in accordance with the present invention may be in a variety of shapes including, but not limited to, amorphous, spherical, crystalline, flat (e.g., membrane), or tubular hydrogels. In a preferred embodiment, the PEG-based insoluble hydrogel is composed of spherical microparticles having a particle size of 1 to 1000 microns, preferably 10 to 100 microns.

PEG-based insoluble hydrogels suitable for disinfection according to the present invention are composed of backbone moieties interconnected by degradable bonds. Optionally, the backbone moieties may be cross-linked by oligomeric, polymeric or low molecular weight cross-linking moieties which are linked to the backbone by degradable bonds and may additionally carry degradable bonds. Optionally, the backbone moiety may carry a bond permanently attached to one or more of the following moieties: ligands, chelating groups, spacer molecules, blocking groups.

In one embodiment of the invention, the hydrogel has the following composition.

The biodegradable PEG-based insoluble hydrogel is composed of backbone moieties interconnected by hydrolytically degradable bonds. Preferably, the backbone moiety has a molecular weight of 1-20kDa, more preferably 1-15 kDa.

Preferably, in the biodegradable PEG-based insoluble hydrogel, the backbone structure portion is characterized by the number of functional groups thereof, which are composed of the interconnected biodegradable functional groups and reactive functional groups. Preferably the total number of interconnected biodegradable groups and reactive functional groups is equal to or greater than 16, preferably 16 to 128, preferably 20 to 100, still preferably 20 to 40, more preferably 24 to 80, still more preferably 28 to 32, even more preferably 30 to 60, most preferably 30 to 32. It is understood that in addition to the interconnecting functional group and the reactive functional group, a protecting group may be present.

The functional groups may be linked to a linear chain. In this case, the functional groups may be regularly or irregularly or alternately spaced in the chain, which may be partially terminated by two dendritic structures, thereby providing the complete functionality.

Preferably, the backbone moiety is characterized by a branched core from which at least 3 PEG-based polymer chains extend. Such branched cores may include polyalcohols or oligoalcohols in bonded form, preferably pentaerythritol, tripentaerythritol, hexaglycerol, sucrose, sorbitol, fructose, mannitol, glucose, cellulose, amylose, starch, hydroxyalkyl starch, polyvinyl alcohol, dextran, hyaluronic acid; or the branched core may comprise a polyamine or oligoamine in bonded form, such as ornithine, diaminobutyric acid, trilysine, tetralysine, pentalysine, hexalysine, heptalysine, octalysine, nonalysine, decalysine, undecalysine, dodecalysine, tridecysine, tetradecysine, pentadecalysine or oligolysine, polyethyleneimine, polyvinylamine. Preferred branched cores may comprise polyamines or oligoamines in bonded form, such as trilysine, tetralysine, pentalysine, hexalysine, heptalysine, octalysine, nonalysine, decalysine, undecalysine, dodecalysine, tridecysine, tetradecysine, pentadecalysine or oligolysine, polyethyleneimines, polyvinylamines.

Preferably the branched core extends 3 to 16 PEG-based polymer chains, more preferably 4 to 8.

The total number of interconnecting functional groups and reactive functional groups of the backbone moiety is divided equally by the number of PEG-based polymer chains extending from the branched core. If the number of PEG-based polymer chains extending from the branched core does not allow for even distribution, it is preferred that the deviation from the average of the number of interconnecting and reactive functional groups per PEG-based polymer chain be kept to a minimum.

More preferably, the total number of interconnecting functional groups and reactive functional groups of the backbone moiety is divided equally by the number of PEG-based polymer chains extending from the branched core. For example, if there are 32 interconnecting functional groups and reactive functional groups, each of the 4 PEG-based polymer chains extending from the core may have 8 groups, preferably via a dendritic moiety attached to the end of each PEG-based polymer chain. Alternatively, each of the 8 PEG-based polymer chains extending from the core may have 4 groups, or each of the 16 PEG-based polymer chains has 2 groups.

For the corresponding PEG-based polymer chains extending from the branched core, structures suitable for the backbone moiety are preferably multi-arm PEG-based derivatives, described in detail, for example, in JenKem Technology, a product listing in the united states (visited by download at www.jenkemusa.com, 28/7 2009), 4-arm PEG derivatives (pentaerythritol core), 8-arm PEG derivatives (hexaglycerol core), and 8-arm PEG derivatives (tripentaerythritol core). Most preferred are 4-arm PEG amine (pentaerythritol core) and 4-arm PEG carboxyl (pentaerythritol core), 8-arm PEG amine (hexaglycerol core), 8-arm PEG carboxyl (hexaglycerol core), 8-arm PEG amine (tripentaerythritol core) and 8-arm PEG carboxyl (tripentaerythritol core). The molecular weight of the multi-arm PEG derivative in the backbone moiety is preferably 1-20kDa, more preferably 2.5-15kDa, even more preferably 5-10 kDa. It will be appreciated that these agents are present in the hydrogel in a bound form.

The additional functional groups may be provided by dendritic structural moieties. Preferably, each dendritic moiety has a molecular weight of from 0.4 to 4kDa, more preferably from 0.4 to 2 kDa. Preferably each dendritic moiety has at least 3 branches and at least 4 reactive functional groups, and at most 63 branches and 64 reactive functional groups; preferably at least 7 branches and at least 8 reactive functional groups, and at most 31 branches and 32 reactive functional groups.

Examples of such dendritic moieties are trilysine, tetralysine, pentalysine, hexalysine, heptalysine, octalysine, nonalysine, decalysine, undecenyl lysine, dodecalysine, tridecysine, tetradecysine, pentadecalysine, hexadecysine, heptadecalysine, octadecalysine, nonadecanonalysine in bonded form. Examples of such preferred dendritic moieties include trilysine, tetralysine, pentalysine, hexalysine, heptalysine in bonded form, most preferably trilysine, pentalysine or heptalysine in bonded form.

Most preferably, the biodegradable PEG-based insoluble hydrogel is characterized by a backbone moiety of the formula C (A-Hyp)₄Wherein each a is independently a polyethylene glycol-based polymer chain, the ends of said polymer chain being linked to the quaternary carbon by permanent covalent bonds, and the distal ends of said PEG-based polymer chain being covalently bonded to a dendritic structural moiety Hyp, wherein each dendritic structural moiety Hyp has at least 4 biodegradable functional groups, reactive functional groups and permanent functional groups that are interconnectedFunctional groups of the bond. Each backbone moiety contains at least 16 interconnected biodegradable functional groups, reactive functional groups and permanent bonds, preferably 20-64, more preferably 28-64 interconnected biodegradable functional groups, reactive functional groups and permanent bonds.

Preferably each A is independently selected from the formula- (CH)₂)_n1(OCH₂CH₂)_nX-wherein n1 is 1 or 2; n is an integer of 5 to 50; and X is a functional group covalently linking a to Hyp.

Preferably, a and Hyp are covalently linked through an amide functional group.

Preferably, the dendritic structural moiety Hyp is a hyperbranched polypeptide. Preferably, the hyperbranched polypeptide is composed of lysine in bonded form, most preferably Hyp is undelysyl or heptalysyl. Preferably, each of the dendritic structural moieties Hyp has a molecular weight of from 0.4 to 4 kD. It is understood that the backbone moiety C (A-Hyp)₄May be composed of the same or different dendritic structural moieties Hyp, and each Hyp may be independently selected. Each moiety Hyp is composed of 5 to 21 lysines, preferably at least 7 lysines, i.e. each moiety Hyp is composed of 5 to 32 lysines in bonded form, preferably at least 7 lysines in bonded form.

Preferably C (A-Hyp)₄Has a molecular weight of 1-20kDa, more preferably 1-15kDa, even more preferably 1-10 kDa.

The biodegradability of the hydrogels of the present invention is achieved by introducing hydrolyzable degradation bonds.

Preferably, the backbone moieties may be linked together by crosslinker moieties, wherein each crosslinker moiety is terminated by at least 2 hydrolytically degradable bonds. In addition to the capped degradable bonds, the crosslinker moieties may also comprise other biodegradable bonds. Thus, each end of the crosslinker moiety linked to the backbone moiety comprises a hydrolytically degradable bond, and additional biodegradable bonds may optionally be present in the crosslinker moiety.

Thus, the biodegradable PEG-based insoluble hydrogel comprises backbone moieties interconnected by hydrolytically degradable bonds, wherein the backbone moieties are preferably linked together by crosslinker moieties, each crosslinker moiety being terminated by at least 2 hydrolytically degradable bonds.

The biodegradable PEG-based insoluble hydrogel may comprise 1 or more different types of crosslinker moieties, preferably 1. The crosslinker moiety may be a linear or branched molecule, and is preferably a linear molecule. In a preferred embodiment of the invention, the crosslinker moiety is linked to the backbone moiety by at least 2 biodegradable bonds.

The term "biodegradable linkage" describes a linkage that is hydrolytically non-enzymatically degradable under physiological conditions (aqueous buffer at pH7.4, 37 ℃) in a half-life of 1 hour to 3 months, including but not limited to aconityl, acetal, carboxylic anhydride, carboxylic ester, imine, hydrazone, maleamic acid amide, orthoester, phosphoramide, phosphate ester, phosphosil ester, silyl ester, sulfonate ester, aromatic carbamate, combinations thereof, and the like. Preferred biodegradable linkages are carboxylate, carbonate, phosphate and sulfonate carboxylates, most preferably carboxylate or carbonate.

Preferably the crosslinker moiety has a molecular weight of 60Da to 5kDa, more preferably 60Da to 4kDa, even more preferably 60Da to 3kDa, even more preferably 0.5-4kDa, even more preferably 1-4kDa, most preferably 1-3 kDa. In one embodiment, the crosslinker moiety is comprised of a polymer.

In addition to oligomeric or polymeric crosslinking moieties, low molecular weight crosslinking moieties may be used, especially when hydrophilic high molecular weight backbone moieties are used to form the biodegradable PEG-based insoluble hydrogels.

Preferably the polyethylene glycol based crosslinker moieties are hydrocarbon chains comprising ethylene glycol units, optionally comprising other functional groups, wherein the polyethylene glycol based crosslinker moieties each comprise at least m ethylene glycol units, wherein m is an integer from 3 to 100, preferably from 1 to 70, most preferably from 10 to 70. Preferably the polyethylene glycol based cross-linking agent moiety has a molecular weight of from 60Da to 5kDa, more preferably from 0.5 to 5 kDa.

Preferably, the crosslinker moiety is a PEG group, preferably only one PEG group molecular chain. Preferably the polyethylene glycol based crosslinker moieties are hydrocarbon chains comprising 1 or more ethylene glycol units, optionally comprising other chemical functional groups, wherein the polyethylene glycol based crosslinker moieties each comprise at least m ethylene glycol units, wherein m is an integer from 1 to 100, preferably from 3 to 100, preferably from 1 to 70, even more preferably from 10 to 70. Preferably the polyethylene glycol based cross-linking agent has a molecular weight of 60Da to 5kDa, preferably 0.5-5 kDa.

In a preferred embodiment of the invention, the crosslinker moiety is constituted by a PEG chain, which is symmetrically linked by ester bonds to 2 α, ω -aliphatic dicarboxylic acid spacers provided by a backbone moiety linked by permanent amide bonds to a hyperbranched dendritic moiety.

The dicarboxylic acid of the spacer moiety which is attached to the backbone moiety and on the other hand to the crosslinking moiety consists of 3 to 12 carbon atoms, most preferably 5 to 8 carbon atoms, and may be substituted on one or more carbon atoms. Preferred substituents are alkyl, hydroxy, amido or substituted amino. The methylene groups of one or more aliphatic dicarboxylic acids may be optionally substituted with O or NH or alkyl substituted N. Preferred alkyl groups are linear or branched alkyl groups having 1 to 6 carbon atoms.

The rate of hydrolysis of the biodegradable bond between the backbone moiety and the crosslinker moiety is influenced or determined by the number and type of linking atoms adjacent to the carboxyl groups of the PEG ester. For example, the degradation half-life of the biodegradable PEG-based insoluble hydrogel can be altered by selecting succinic acid, adipic acid, or glutaric acid to form PEG esters.

In another embodiment, a multifunctional moiety is attached to the reactive functional groups of the polymerized hydrogel to increase the number of reactive functional groups, which may, for example, increase the drug loading of the biodegradable PEG-based insoluble hydrogel. Such multifunctional moieties may be provided by derivatives of lysine, dilysine, trilysine, tetralysine, pentalysine, hexalysine, heptalysine or oligolysine, low molecular weight PEI in bonded form and appropriately substituted. Preferably, the multifunctional moiety is composed of lysine in bonded form. Such multifunctional moieties may optionally be protected with a protecting group.

In addition, such hydrogels of the invention may be functionalized with spacers bearing the same functional groups, for example, by incorporating hetero-bifunctional spacers, such as appropriately activated COOH-PEG₆-NH-Fmoc, and removing the Fmoc protecting group to introduce an amino group into the hydrogel.

One preferred crosslinker moiety is shown below; the dotted line represents a biodegradable bond interconnecting the backbone moieties:

wherein q is an integer from 5 to 50.

Preferably, the PEG-based insoluble hydrogel is composed of backbone moieties interconnected by hydrolytically degradable bonds.

More preferably, the backbone moiety comprises a branched core of the formula:

wherein the dashed line indicates a link to the remainder of the backbone moiety.

More preferably, the backbone moiety comprises the formula:

wherein n is an integer from 5 to 50 and the dashed line indicates a link to the remainder of the backbone moiety.

Preferably the backbone moiety comprises the hyperbranched moiety Hyp.

More preferably, the backbone moiety comprises a hyperbranched moiety Hyp of the formula:

wherein the dotted line indicates the attachment to the rest of the molecule, the carbon atom marked with an asterisk indicates the S-configuration in a preferred embodiment. However, it is understood that the hyperbranched moiety Hyp as shown above may also be in the R-configuration or may be a racemate.

Preferably, the backbone moiety is linked to at least one spacer of the formula:

one of the dotted lines is linked to the hyperbranched moiety Hyp, the second dotted line represents the linkage to the rest of the molecule; and wherein m is an integer from 2 to 4.

Preferably the backbone moieties are linked together by a crosslinker moiety having the structure:

wherein q is an integer from 3 to 100.

More preferably, the backbone moieties of the PEG-based insoluble hydrogel are bonded together by moieties of the formula:

wherein each dashed line represents a link to a backbone moiety, respectively, and wherein n is 45.

Still more preferably, the backbone moieties of the PEG-based insoluble hydrogel are bonded together by moieties of the formula:

wherein the dashed lines each represent a link to a backbone moiety, and wherein n is 22.

The present invention describes the disinfection of biodegradable PEG-based insoluble hydrogels by irradiation in the presence of protective solvents. The biodegradable PEG-based insoluble hydrogel used in this disinfection procedure is solvated with a protective solvent prior to irradiation and the protective solvent remains present during irradiation. The protective solvent is preferably selected from acetic acid (aqueous solution, 0.01-1% (v/v)), acetonitrile, 4-acetylmorpholine, Dimethylsulfoxide (DMSO), Dichloromethane (DCM), N-Dimethylacetamide (DMA), N-Dimethylformamide (DMF), 1, 3-dimethyl-2-imidazolidinone (DMI), dimethyl carbonate, dimethylformamide, 1-ethyl-2-pyrrolidone, N-ethylacetamide, N-ethylformamide, formamide, 4-formylmorpholine, 1-formylpyrrolidone, 1, 3-dimethyl-2-imidazolidinone, 1, 3-dimethyl-3, 4,5, 6-tetrahydro-2 (1H) -pyrimidinone (DMPU), alkyl alcohols such as methanol, ethanol, propanol; formamide, Hexamethylphosphoramide (HMPA), N-methylacetamide, nicotinamide (aqueous solution, 0.1-5% (w/w)), pyridoxine (aqueous solution, 0.1-5% (w/w)), N-methylformamide, NMP, propylene 1, 2-carbonate, Tetrahydrofuran (THF), sulfolane, water, or mixtures thereof.

More preferably the protective solvent is selected from acetic acid (aqueous solution, 0.01-1% (v/v)), acetonitrile, Dimethylsulfoxide (DMSO), Dichloromethane (DCM), Dimethylacetamide (DMA), dimethyl carbonate, dimethylformamide, 1, 3-dimethyl-2-imidazolidinone, 1, 3-dimethyl-3, 4,5, 6-tetrahydro-2 (1H) -pyrimidinone (DMPU), alkyl alcohols such as methanol, ethanol, propanol; formamide, Hexamethylphosphoramide (HMPA), nicotinamide (aqueous solution, 0.1-5% (w/w)), N-methylformamide, NMP, Tetrahydrofuran (THF), sulfolane, water, or mixtures thereof.

Most preferably the protective solvent is selected from 4-acetylmorpholine, DMA, DMF, DMI, DMPU, 1-ethyl-2-pyrrolidone, N-ethylacetamide, N-ethylformamide, 4-formylmorpholine, 1-formylpyrrolidone, N-methylacetamide, N-methylformamide, DMSO or NMP. Even more preferably, the protective solvent is selected from DMSO, DMA, DMF, DMI, or NMP; even more preferably DMA, DMF, DMI or NMP; still even more preferred is DMSO or NMP, even more preferred is NMP.

The protective solvent is optionally degassed and may optionally contain other substances dissolved in the protective solvent, such as one or more protective agents, such as salts. Preferably, the protective agent is included at a concentration of 0.01-10%. It is to be understood that the protective solvent may also be a mixture of two or more protective solvents or an aqueous dilution thereof.

The protective agent may be chosen from optionally substituted linear, branched or cyclic C₁-C₁₀An alkylamine; optionally substituted, linear or branched C₁-C₁₀An alkyl carboxylic acid; optionally substituted, linear or branched C₁-C₁₀An alkyl sulfonic acid; optionally substituted, linear or branched C₁-C₁₀Alkyl thiols or carbohydrates. The protective agent may be substituted with a hydroxyl group or substituted with or interrupted by a urea, amide or carbamate group or interrupted by an ether. A mixture of two or more protective agents may be added to the protective solvent.

Preferably, the protective agent is selected from propylamine, butylamine, pentylamine, sec-butylamine, ethanolamine, diethanolamine, serinol, tris (hydroxymethyl) aminomethane, acetic acid, formic acid, ascorbic acid, glycinamide, pivalic acid, propionic acid, succinic acid, glutaric acid, adipic acid, thioglycerol, dithiothreitol, mercaptoethanol, reduced glutathione.

For sterilization, the biodegradable PEG-based insoluble hydrogel is placed in a suitable container, which ensures sterility after the sterilization procedure. Thus, the hydrogel is solvated with a protective solvent, the appropriate container is closed and the sterilization process is carried out. The appropriate container is selected to ensure sterility after closing the container and performing the sterilization procedure. Alternatively, the PEG-based insoluble hydrogel is first solvated with a protective solvent and then transferred to a suitable container where sterilization is performed, and then the suitable container is closed. The sterilization of the biodegradable PEG-based insoluble hydrogel is performed by irradiation, preferably with gamma irradiation at a dose of 5-100kGy, preferably 8-50kGy, more preferably 20-40kGy such as 32-40kGy, more preferably 20-30 kGy. Irradiation of the biodegradable PEG-based insoluble hydrogels of the present invention can be performed at a temperature of room temperature (25 ℃) to-80 ℃. Preferably, the irradiation is carried out at room temperature. To achieve temperatures below room temperature, a suitable container containing the biodegradable PEG-based insoluble hydrogel to be sterilized may be stored in a coolable environment or may be surrounded by a cooling substance (e.g., ice or dry ice).

Such sterilized biodegradable PEG-based insoluble hydrogels can be used directly, e.g., as implants, or can be further modified, e.g., by conjugating a bioactive moiety to the sterilized biodegradable PEG-based insoluble hydrogel. In the latter case, further processing is performed under sterile conditions using pre-sterilization chemicals and biologically active moieties.

In one embodiment of the invention, the small molecule bioactive moiety is conjugated to a functional group of the biodegradable PEG-based insoluble hydrogel, thereby obtaining a so-called biodegradable PEG-based insoluble hydrogel with small molecule bioactive moieties, which is then sterilized by irradiation in the presence of a protective solvent. It will be apparent to those skilled in the art that only the biologically active moieties of such small molecules are suitable, which retain their chemical structure during gamma irradiation.

Accordingly, a preferred aspect of the invention is the method of the invention, wherein the biodegradable PEG-based insoluble hydrogel is loaded with a small molecule bioactive moiety.

If the biodegradable PEG-based insoluble hydrogel is loaded with a small molecule bioactive moiety and sterilized by irradiation by the method of the invention, the small molecule bioactive moiety should be retained. In the context of the present invention, the term "retain" preferably means that at least 90% of the biologically active moieties of the small molecules released from the sterilized hydrogel are not altered, as can be determined by methods known to those skilled in the art, such as by mass spectrometry, ultra-high performance liquid chromatography or pharmacological activity assays.

In a preferred embodiment of the present invention, the dried biodegradable PEG-based insoluble hydrogel is solvated using 5-10ml NMP per g of dried biodegradable PEG-based insoluble hydrogel and irradiated with gamma rays at a dose of 25kGy, using a closed container to prevent contamination after sterilization. For further processing, NMP is replaced with the desired solvent using a syringe equipped with a filter or suitable column, similar to binding the bioactive moiety to the sterilized hydrogel. It is obvious to those skilled in the art that all steps after sterilization of the biodegradable PEG-based insoluble hydrogel are performed under aseptic conditions using sterile solutions.

In an even more preferred embodiment of the present invention, the biodegradable PEG-based insoluble hydrogel is solvated using 5 to 10ml NMP containing 0.1 to 2% (v/v) ethanolamine or propylamine per 1g of the dried biodegradable PEG-based insoluble hydrogel, followed by irradiation of the solvated biodegradable PEG-based insoluble hydrogel with gamma rays at a dose of 32kGy, wherein a closed container is used to prevent contamination after sterilization. For further processing, NMP containing 0.1-2% (v/v) ethanolamine or propylamine is replaced with the desired solvent using a syringe equipped with a filter or suitable column, similar to binding the bioactive moieties to a sterilized hydrogel. It is obvious to those skilled in the art that all steps after sterilization of the biodegradable PEG-based insoluble hydrogel are performed under aseptic conditions using sterile solutions.

Another aspect of the present invention is a sterilized biodegradable PEG-based insoluble hydrogel, in particular a sterilized biodegradable PEG-based insoluble hydrogel loaded with a small molecule bioactive moiety, obtainable by any of the methods of the present invention.

FIG. 1 shows the in vitro degradation kinetics at pH9.0, 37 ℃ for 4a (FIG. 1a), 4b (FIG. 1b), 4C (FIG. 1C), 4d (FIG. 1d) and 4e (FIG. 1e) after sterilization, respectively, together with 3a (unsterilized hydrogel; "control"), and 4f (FIG. 1f), 4g (FIG. 1g), 4h (FIG. 1h), 4i (FIG. 1i), 4j (FIG. 1j) and 4k (FIG. 1k) after sterilization, respectively, with 3b (unsterilized hydrogel; "control") at pH9.0, 37 ℃.

Figure 2 shows the in vitro degradation kinetics of 6 after sterilization together with 5 (non-sterilized hydrogel; "control") at ph9.0, 37 ℃.

Examples

Materials and methods

Material

Amino 4-arm PEG5000 was obtained from JenKem Technology, beijing, china.

All other chemicals were obtained from Sigma-ALDRICH Chemie GmbH, Taufkirchen, Germany.

In the case of hydrogel beads, a syringe equipped with a polypropylene frit was used as a reaction vessel or for a washing step.

And (3) analysis:

electrospray ionization mass spectrometry (ESI-MS) was performed on a Thermo Fisher Orbitrap Discovery instrument equipped with a Waters acquisition UPLC system.

Due to the polydispersity of the PEG starting material, the MS spectra of the PEG products show a series of (A), (B), (C), (CH₂CH₂O)_nA structural portion. For easier illustration, only one single representative m/z signal is given in the examples.

If not otherwise stated, Size Exclusion Chromatography (SEC) was performed using an Amersham Bioscience AEKTAbasic system (Amersham Bioscience/GE Healthcare) equipped with Superdex755/150GL columns. 4/1(v/v) aqueous buffer (20mM sodium phosphate, 150mM NaCl, 0.005% TWEEN 20, pH 7.4)/acetonitrile mixture was used as the mobile phase. Absorption was detected at 215 nm.

Example 1

Synthesis of backbone reagent 1g

Backbone reagent 1g was synthesized from amino 4-arm PEG50001a according to the following protocol:

to synthesize compound 1b, 4-arm PEG5000 tetramine 1a (MW approx. 5200g/mol, 5.20g, 1.00mmol, HCl salt) was dissolved in 20mL DMSO (anhydrous). Boc-Lys (Boc) -OH (2.17g, 6.25mmol), EDC HCl (1.15g, 6.00mmol), HOBt. H in 5mL DMSO (anhydrous) were added₂O (0.96g, 6.25mmol) and collidine (5.20mL, 40 mmol). The reaction mixture was stirred at RT for 30 min.

The reaction mixture was diluted with 1200mL of dichloromethane and 600mL of 0.1N H₂SO₄(2X), brine (1X), 0.1M NaOH (2X) and 1/1(v/v) brine/water (4X). The aqueous layer was back-extracted with 500mL DCM. The organic phase is passed through Na₂SO₄Dried, filtered and evaporated to yield 6.3g of crude product 1b as colorless oil. Compound 1b was purified by RP-HPLC.

Yield: 3.85g (59%) of the colorless glassy product 1 b.

MS：m/z 1294.4=[M+5H]⁵⁺(calculated = 1294.6).

Compound 1c was prepared by dissolving 3.40g of compound 1b (0.521mmol) in 5mL of methanol and in two9mL 4N HCl in alkane was obtained at RT with stirring for 15 min. Volatiles were removed in vacuo. The product was used in the next step without further purification.

MS：m/z 1151.9=[M+5H]⁵⁺(calculated = 1152.0).

To synthesize compound 1d, 3.26g of compound 1c (0.54mmol) were dissolved in 15mL of DMSO (anhydrous). 2.99g of Boc-Lys (Boc) -OH (8.64mmol), 1.55g of EDC HCl (8.1mmol), 1.24g of HOBt. H in 15mL of DMSO (anhydrous) were added₂O (8.1mmol) and 5.62mL collidine (43 mmol). The reaction mixture was stirred at RT for 30 min.

The reaction mixture was diluted with 800mL of DCM and 400mL of 0.1N H₂SO₄(2X), brine (1X), 0.1M NaOH (2X) and 1/1(v/v) brine/water (4X). The aqueous layer was back-extracted with 800mL DCM. The organic phase is passed through Na₂SO₄Dried, filtered and evaporated to obtain a glassy crude product.

The product was dissolved in DCM and precipitated with cooled (-18 ℃) ether. This procedure was repeated twice and the precipitate was dried in vacuo.

Yield: 4.01g (89%) of the colorless glassy product 1d were used in the next step without further purification.

MS：m/z 1405.4=[M+6H]⁶⁺(calculated = 1405.4).

Compound 1e was prepared by mixing compound 1d (3.96g, 0.47mmol) in 7mL of methanol with compound 1d in twoA solution of 20mL 4N HCl in an alkane was obtained at RT with stirring for 15 min. Volatiles were removed in vacuo. The product was used in the next step without further purification.

MS：m/z 969.6=[M+7H]⁷⁺(calculated = 969.7).

To synthesize compound 1f, compound 1e (3.55g, 0.48mmol) was dissolved in 20mL of DMSO (anhydrous). Boc-Lys (Boc) -OH (5.32g, 15.4mmol), EDC HCl (2.76g, 14.4mmol), HOBt. H in 18.8mL DMSO (anhydrous) were added₂O (2.20g, 14.4mmol) and 10.0mL collidine (76.8 mmol). The reaction mixture was stirred at RT for 60 min.

The reaction mixture was diluted with 800mL of DCM and 400mL of 0.1N H₂SO₄(2X), brine (1X), 0.1M NaOH (2X) and 1/1(v/v) brine/water (4X). The aqueous layer was back-extracted with 800mL DCM. The organic phase is passed through Na₂SO₄Dried, filtered and evaporated to give the crude product 1f as a colorless oil.

The product was dissolved in DCM and precipitated with cooled (-18 ℃) ether. This procedure was repeated twice and the precipitate was dried in vacuo.

Yield: 4.72g (82%) of the colorless glassy product 1f were used in the next step without further purification.

MS：m/z 1505.3=[M+8H]⁸⁺(calculated = 1505.4).

Backbone reagent 1g was prepared by mixing compound 1f (MW about 12035g/mol, 4.72g, 0.39mmol) in 20mL of methanol and in twoA solution of 40mL 4N HCl in an alkane was obtained at RT with stirring for 30 min. Volatiles were removed in vacuo.

Yield: 3.91g (100%) glassy product backbone reagent 1 g.

MS：m/z 977.2=[M+9H]⁹⁺(calculated =977.4)。

Alternative Synthesis route to 1g

To synthesize compound 1b, boc-Lys (boc) -Osu (26.6g, 60.0mmol) and DIEA (20.9mL, 120mmol) were added to a suspension of 4-arm PEG5000 tetramine (1a) (50.0g, 10.0mmol) at 45 ℃ and in 250mL iPrOH (anhydrous), and the mixture was stirred for 30 min.

N-propylamine (2.48mL, 30.0mmol) was then added. After 5 minutes, the solution was diluted with 1000mL MTBE and stored overnight at-20 ℃ without stirring. Approximately 500mL of supernatant was decanted and discarded. 300mL of cold MTBE was added, and after shaking for 1 minute the product was collected by filtration through a glass filter and washed with 500mL of cold MTBE. The product was dried under vacuum for 16 hours.

Yield: 65.6g (74%) of a white lumpy solid 1b

MS：m/z=937.4=[M+7H]⁷⁺(calculated = 937.6).

Compound 1C was obtained by stirring compound 1b (48.8g, 7.44mmol) from the previous step in 156mL 2-propanol at 40 ℃. A mixture of 196mL of 2-propanol and 78.3mL of acetyl chloride was added over 1-2 minutes with stirring. The solution was stirred at 40 ℃ for 30 minutes and allowed to cool to-30 ℃ overnight without stirring. 100mL of cold MTBE was added, the suspension was shaken for 1 minute and allowed to cool at-30 ℃ for 1 hour. The product was collected by filtration through a glass filter and washed with 200mL of cold MTBE. The product was dried under vacuum for 16 hours.

Yield: 38.9g (86%) of 1c in the form of a white powder

MS：m/z=960.1[M+6H]⁶⁺(calculated = 960.2).

To synthesize compound 1d, boc-Lys (boc) -OSu (16.7g, 37.7mmol) and DIEA (13.1mL, 75.4mmol) were added to a suspension of 1C from the previous step (19.0g,3.14mmol) at 45 ℃ in 80mL 2-propanol and the mixture was stirred at 45 ℃ for 30 min. N-propylamine (1.56mL, 18.9mmol) was then added. After 5 minutes, the solution was precipitated with 600mL of cold MTBELiquid and centrifuge (3000 min)^-11 minute). The precipitate was dried in vacuo for 1 hour and dissolved in 400mL THF. 200mL of diethyl ether was added and the product was cooled to-30 ℃ for 16 hours without stirring. The suspension was filtered through a glass filter and washed with 300mL of cold MTBE. The product was dried under vacuum for 16 hours.

Yield: 21.0g (80%) of white powdery 1d

MS：m/z 1405.4=[M+6H]⁶⁺(calculated = 1405.4).

Compound 1e was obtained by dissolving compound 1d (15.6g, 1.86mmol) obtained from the previous step in 3N HCl in methanol (81mL, 243mmol) and stirring for 90 min at 40 ℃. 200mL MeOH and 700mL iPrOH were added and the mixture was stored at-30 ℃ for 2 hours. For complete crystallization, 100mL MTBE was added and the suspension was stored overnight at-30 ℃. 250mL of cold MTBE was added, the suspension was shaken for 1 min and filtered through a glass filter, washed with 100mL of cold MTBE. The product was dried in vacuo.

Yield: 13.2g (96%) of 1e in the form of a white powder

MS：m/z=679.1=[M+10H]¹⁰⁺(calculated = 679.1).

To synthesize compound 1f, to a suspension of 1e from the previous step (8.22g, 1.12mmol) at 45 ℃ in 165mL 2-propanol was added boc-Lys (boc) -OSu (11.9g, 26.8mmol) and DIEA (9.34mL, 53.6mmol), and the mixture was stirred at 45 ℃ for 30 min. N-propylamine (1.47mL, 17.9mmol) was then added. After 5 minutes, the solution was cooled to-18 ℃ for 2 hours, then 165mL of cold MTBE was added, the suspension was shaken for 1 minute and filtered through a glass filter. The filter cake was then washed with 4X 200mL cold MTBE/iPrOH (4:1) and 1X 200mL cold MTBE. The product was dried under vacuum for 16 hours.

Yield: 12.8g of a pale yellow block solid MW (90%)1f

MS：m/z 1505.3=[M+8H]⁸⁺(calculated = 1505.4).

Backbone reagent 1g was prepared by coupling 4-arm PEG5kDa (-LysLys)₂Lys₄(boc)₈)₄(1f) (15.5g, 1.29mmol) was dissolved in 30mL MeOH and cooled to 0 ℃. Add in two within 3 minutes4N HCl in alkane and remove ice bath. After 20 min, 3N HCl in methanol (200mL, 600mmol, cooled to 0 ℃) was added over 15 min and the solution was stirred at RT for 10 min. The product solution was precipitated with 480mL of cold MTBE and centrifuged at 3000rpm for 1 minute. The precipitate was dried under vacuum for 1 hour and redissolved in 90mL MeOH, precipitated with 240mL cold MTBE, and the suspension was centrifuged again at 3000rpm for 1 minute. The product was dried in vacuo.

Yield: 11.5g (89%), pale yellow flakes.

MS：m/z=1104.9[M+8H]⁸⁺(calculated = 1104.9).

Example 2

Synthesis of crosslinker reagent 2d

Crosslinker reagent 2d was prepared from monobenzyladipate (English, Arthur R. et al, Journal of Medicinal Chemistry, 1990, 33(1), 344-:

a solution of PEG2000(2a) (11.0g, 5.5mmol) and benzyl half ester of adipic acid (4.8g, 20.6mmol) in dichloromethane (90.0mL) was cooled to 0 ℃. Dicyclohexylcarbodiimide (4.47g, 21.7mmol) was added followed by a catalytic amount of DMAP (5mg) and the solution was stirred and allowed to reach RT overnight (12 h). The flask was stored at +4 ℃ for 5 hours. The solid was filtered and the solvent was completely removed by vacuum distillation. The residue was dissolved in 1000mL 1/1(v/v) ether/ethyl acetate and stored at RTThe reaction was allowed to stand for 2 hours while forming a small amount of flaky solid. Passing said solid throughThe pad was filtered off. The solution was stored in a closed flask at-30 ℃ in a freezer for 12 hours until crystallization was complete. The crystallized product was filtered through a frit and washed with cooled ether (-30 ℃). The filter cake was dried under vacuum.

Yield: 11.6g (86%) 2b as a colorless solid. The product was used in the next step without further purification.

MS：m/z 813.1=[M+3H]³⁺(calculated =813.3)

In a 500mL glass autoclave, PEG2000 dibenzyl bis adipate 2b (13.3g, 5.5mmol) was dissolved in ethyl acetate (180mL) and 10% palladium on charcoal (0.4g) was added. The solution was hydrogenated at 6 bar and 40 ℃ until hydrogen consumption ceased (5-12 hours). By passingThe catalyst was removed by pad filtration and the solvent evaporated in vacuo.

Yield: 12.3g (quantitative) of 2c as yellow oil. The product was used in the next step without further purification.

MS：m/z 753.1=[M+3H]³⁺(calculated =753.2)

A solution of PEG2000 bis-adipate half ester 2c (9.43g, 4.18mmol), N-hydroxysuccinimide (1.92g, 16.7mmol) and dicyclohexylcarbodiimide (3.44g, 16.7mmol) in 75mL DCM (anhydrous) was stirred at RT overnight. The reaction mixture was cooled to 0 ℃ and the precipitate was filtered off. DCM was evaporated and the residue was recrystallized from THF.

Yield: 8.73g (85%) of a colorless solid crosslinker reagent 2d

MS：m/z 817.8=[M+3H]³⁺(calculated = 817.9).

Example 3

Preparation of amino-free Low-Density hydrogel beads 3a

A solution of 300mg 1g and 900mg 2d in 10.80mL DMSO was added to a solution of 100mg Arlacel P135(Croda International Plc) in 80mL heptane. The mixture was stirred at 700rpm for 10 minutes at RT with a custom metal stirrer to form a suspension. 1.1mL of N, N, N ', N' -Tetramethylenediamine (TMEDA) was added to conduct polymerization. After 2 hours, the stirrer speed was reduced to 400rpm and the mixture was stirred for a further 16 hours. Acetic acid (1.6 mL) was added, followed by 50mL of water after 10 minutes. After 5 minutes, the stirrer was stopped and the aqueous phase was drained.

For bead size classification, the water-hydrogel suspensions were wet sieved on 75 μm, 50 μm, 40 μm, 32 μm and 20 μm steel sieves. The bead fractions remaining on the 32 μm, 40 μm and 50 μm sieves were collected and washed 3 times with water, 10 times with ethanol and dried at 0.1 mbar for 16 hours to obtain 3a as a white powder.

Preparation of amino-free Medium Density hydrogel beads 3b

A solution of 1200mg 1g and 3840mg 2d in 28.6mL DMSO was added to a solution of 425mg Arlacel P135(Croda International Plc) in 100mL heptane. The mixture was stirred at 650rpm for 10 minutes at RT with a custom metal stirrer to form a suspension. 4.3mL of N, N, N ', N' -Tetramethylenediamine (TMEDA) was added to conduct polymerization. After 2 hours, the stirrer speed was reduced to 400rpm and the mixture was stirred for a further 16 hours. Acetic acid (6.6 mL) was added, followed by water (50 mL) after 10 minutes. After 5 minutes, the stirrer was stopped and the aqueous phase was drained.

To size the beads, the water-hydrogel suspension was wet sieved on 63 μm, 50 μm, 40 μm, 32 μm and 20 μm steel sieves. The bead fractions retained on the 32 μm, 40 μm and 50 μm sieves were collected and washed 3 times with water, 10 times with ethanol and dried at 0.1 mbar for 16 hours, yielding 2.86g of 3b as a white powder.

Preparation of amino-free high-Density hydrogel beads 3c

A solution of 2400mg 1g and 3600mg 2d in 24.0mL DMSO was added to a solution of 425mg Arlacel P135(Croda International Plc) in 110mL heptane. The mixture was stirred at 850rpm for 10 minutes at RT with a custom metal stirrer to form a suspension. 8.6mL of N, N, N ', N' -Tetramethylenediamine (TMEDA) was added to conduct polymerization. After 2 hours, the stirrer speed was reduced to 400rpm and the mixture was stirred for a further 16 hours. Acetic acid (13.2 mL) was added, followed by water (50 mL) after 10 minutes. After 5 minutes, the stirrer was stopped and the aqueous phase was drained.

To size the beads, the water-hydrogel suspension was wet sieved on 63 μm, 50 μm, 40 μm, 32 μm and 20 μm steel sieves. The bead fractions retained on the 32 μm, 40 μm and 50 μm sieves were collected and washed 3 times with water, 10 times with ethanol and dried at 0.1 mbar for 16 hours, thus obtaining 3.00g of 3c as a white powder.

Example 4 preparation of hydrogel beads and subsequent Gamma irradiation (4a, 4b, 4c, 4d, 4e, 4f, 4g, 4h, 4i, 4j, 4k)

A portion of 20mg of dried hydrogel 3a in a syringe equipped with a filter was washed 5 times with the following protective solvents: NMP (4a), Diethylene Glycol Diethyl Ether (DGDE) (4b), DMSO (4c), or 0.1% acetic acid in water (4 d).

A portion of 20mg of dried hydrogel 3b in a syringe equipped with a filter was also washed 5 times with the following protective solvents: DMI (4f), DMA (4g), NMP +0.5 vol% 1-propylamine (4h), NMP +0.5 vol% 2-ethanolamine (ethanolamine) (4i), NMP +0.1 vol% acetic acid (4j), or NMP (4k) with 0.2M AcOH and 0.1M propylamine.

After the last wash, the syringe was closed, leaving the hydrogel beads in a swollen form with a slight excess of protective solvent.

Further, the dried sample of hydrogel 3a was irradiated in a dried state to provide 4 e.

The samples were gamma-irradiated (irradiation source: Co60) at room temperature with a dose of 40kGy (4a, 4b, 4c, 4d, 4e, 4f) or 32kGy (4g, 4h, 4i, 4j, 4 k). The sample was subsequently washed 5 times with ethanol and dried at 0.1 mbar for 16 hours.

EXAMPLE 5 determination of amino group content

Fmoc-Asp (OtBu) -OSu (49mg, 116. mu. mol) was dissolved in 0.9mL acetonitrile and 0.5mL of 50mM sodium phosphate buffer (pH7.4) was added. This solution was added to 20mg of hydrogels 3a and 4a in a syringe reactor and shaken for 30 minutes at ambient temperature.

The hydrogel was then washed with 10 × acetonitrile/water 2:1(v/v) +0.1% TFA and 10 × DMF.

The Fmoc group was cleaved by shaking with DMF/DBU 98/2(v/v) for 3X 10 min and washing with 10 XDMF/DBU 98/2 (v/v). All these fractions were collected and diluted with DMF and the amount of 9-methylenefluorene was determined by measuring UV absorption at 295 nm. Using 9141L mol^-1cm^-1The extinction coefficient of (a).

Amino group content of 3 a: 0.13mmol/g

Amino content of 4a after gamma irradiation: 0.12mmol/g

The same amino group content values were obtained using Fmoc-Gly-Osu instead of Fmoc-Asp (OtBu) -OSu.

Example 6 accelerated in vitro degradation analysis of hydrogel beads

In vitro degradation kinetics of the sterilized hydrogel beads 4a, 4b, 4C, 4d, 4e, 4f, 4g, 4h, 4i, 4j and 4k and 3a and 3b (control, non-sterilized) under accelerated conditions were measured by incubating 5mg of each sample (3a, 4b, 4C, 4d and 4e) at 37 ℃ in 0.5mL of 0.5M sodium borate buffer (ph 10.3). Alternatively, for accelerating in vitro degradation (3b, 4f, 4g, 4h, 4i, 4j and 4k), 0.5mL of 0.5M sodium borate buffer (pH9.0) at 37 ℃ was used. Aliquots were removed at intervals and analyzed by SEC. The UV signal corresponding to the water-soluble degradation product released from the hydrogel containing one or more backbone moieties was integrated and plotted against the incubation time (figure 1).

Example 7 preparation of hydrogel 6 gamma irradiated and loaded with paliperidone

Paliperidone-loaded hydrogel 5 was prepared by modifying hydrogel 3c with lysine and then combining paliperidone glutaryl ester as described in international patent application PCT/EP 2010/064874. A20 mg portion of the dried hydrogel beads in a syringe equipped with a filter was washed 5 times with formulation buffer (85g/l trehalose dihydrate, 50mM succinate/tris buffer pH5.0, 0.05% Tween 20, 1mM EDTA) and dried 5. After the final washing step, the syringe was closed, leaving the hydrogel beads in swollen form with a slight excess of protective solvent. The sample was gamma-irradiated in a dry ice bed at a dose of 40kGy (irradiation source: Co 60). The hydrogel was subsequently washed 6 times with formulation buffer, 5 times with water and 5 times with ethanol and dried for 16 hours at 0.1 mbar.

Example 8 in vitro degradation of irradiated 6 at pH9 and 37 ℃

The in vitro degradation kinetics of hydrogel 6 and control 5 (unsterilized) under accelerated conditions were measured by incubating 2mg of each sample at 37 ℃ in 1.0mL of 0.5M sodium borate buffer (ph 9.0). Aliquots were removed at regular intervals and analyzed by SEC. The UV signal (215nm) corresponding to the water-soluble degradation product released from a hydrogel comprising one or more backbone moieties was integrated and plotted against the incubation time. Only small deviations in degradation behavior were observed (fig. 2).

EXAMPLE 9 the quality of paliperidone released from irradiated hydrogel 6

1mg of irradiated paliperidone-linker-hydrogel 6 was incubated at 37 ℃ in 1.5mL of phosphate buffer (60mM, 3mM EDTA, 0.01% Tween-20) at pH 7.4. After 4 days, the amount of paliperidone released from the supernatant was determined by using HPLC AWaters Acquity UPLC equipped with a Waters BEH C18 column, 50X 2.1mm I.D., 1.7 μm particle size. Solvent A: 0.05% TFA in water, solvent B: 0.04% TFA in acetonitrile. Within 4 minutes, a linear gradient of 0-50% B was used. Paliperidone (215nm) was obtained at a purity of 95%.

Abbreviations：

AcOEt Ethyl acetate

AcOH acetic acid

Asp aspartic acid

Boc tert-butoxycarbonyl

DBU 1, 8-diazabicyclo [5.4.0] undec-7-ene

DCC dicyclohexylcarbodiimide

DcM Dichloromethane

DGDE diethylene glycol diethyl ether

DMA N, N-dimethylacetamide

DMAP dimethylaminopyridine

DMF N, N-dimethylformamide

DMI 1, 3-dimethyl-2-imidazolidinone

DMSO dimethyl sulfoxide

EDC 1-ethyl-3- (3-dimethylaminopropyl) carboximide

ESI electrospray ionization

eq stoichiometric equivalent

EtOH ethanol

Fmoc fluorenylmethoxycarbonyl

HOBt N-hydroxybenzotriazole

iPrOH Isopropanol

kGy of kilogray

LCMS (liquid Crystal Module System) mass spectrum-liquid chromatography combined application

MeOH methanol

MS Mass Spectrometry

MTBE methyl tert-butyl ether

MW molecular weight

NHS N-hydroxysuccinimide

NMP N-methyl-2-pyrrolidone

OtBu tert-butoxy group

OSu N-hydroxysuccinimide group

PEG polyethylene glycol

PyBOP benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate

RP-HPLC reversed-phase high performance liquid chromatography

RT Room temperature

SEC size exclusion chromatography

tBu tert-butyl

TFA trifluoroacetic acid

THF tetrahydrofuran

TMEDA N, N, N ', N' -tetramethylenediamine

UV ultraviolet ray

VIS vision

Claims (15)

1. A method of disinfecting a biodegradable polyethylene glycol-based insoluble hydrogel comprising backbone moieties interconnected by hydrolytically degradable bonds, the method comprising the steps of:

(a) providing a hydrogel;

(b) solvating the hydrogel in a protective solvent or a mixture of two or more protective solvents or an aqueous solution thereof;

(c) subjecting the solvated hydrogel to gamma irradiation;

wherein the main chain structural partHaving the formula C (A-Hyp)₄Wherein each a is independently a polyethylene glycol-based polymer chain, the ends of said polymer chain being linked to the quaternary carbon by permanent covalent bonds, and the distal ends of the PEG-based polymer chains being covalently bonded to a dendritic structural moiety Hyp, wherein each dendritic structural moiety Hyp is composed of 5-21 lysines;

the main chain moieties are linked together by a cross-linking moiety consisting of a PEG chain symmetrically linked by ester linkages to the 2 α, ω -aliphatic dicarboxylic acid spacers provided by the main chain moieties linked by permanent amide linkages to the hyperbranched dendritic moiety;

the protective solvent is N-methyl-2-pyrrolidone (NMP), N-Dimethylacetamide (DMA), Dimethylformamide (DMF) or 1, 3-dimethyl-2-imidazolidinone (DMI); and may optionally contain one or more protective agents.

2. The method of claim 1, wherein the protective solvent is NMP.

3. The method according to claim 1, wherein the sterilization is obtained by gamma irradiation at a dose of 5-100 kGy.

4. The method of claim 1, wherein the sterilization is obtained by gamma irradiation at a dose of 8-50 kGy.

5. The method of claim 1, wherein the hydrogel is loaded with a small molecule bioactive moiety.

6. The method according to claim 1 or 2, wherein the hydrogel is composed of spherical microparticles having a particle size of 1 to 1000 μm.

7. The method of claim 1, wherein the backbone moieties of the hydrogel each have a molecular weight of 1-20 kDa.

8. The method of claim 1, wherein the crosslinker moiety has a molecular weight of 60Da to 5 kDa.

9. The method according to claim 1, wherein the backbone moiety of the biodegradable PEG-based insoluble hydrogel comprises a hyperbranched moiety Hyp of the formula:

wherein the dotted line indicates the attachment to the rest of the molecule and the carbon atom marked with an asterisk indicates the S-configuration.

10. The method of claim 1 or 2, wherein the backbone moiety is linked to at least one spacer of the formula:

one of the dotted lines indicates the attachment to the hyperbranched moiety Hyp and the second dotted line indicates the attachment to the rest of the molecule; wherein m is an integer from 2 to 4.

11. The method of claim 1, wherein the backbone moieties are bonded together by a crosslinker moiety comprising the structure:

wherein q is an integer from 3 to 100.

12. The method of claim 1 or 2, wherein the protective agent is selected from propylamine, butylamine, pentylamine, sec-butylamine, ethanolamine, diethanolamine, serinol, tris, acetic acid, formic acid, ascorbic acid, glycinamide, pivalic acid, propionic acid, succinic acid, glutaric acid, adipic acid, thioglycerol, dithiothreitol, mercaptoethanol, reduced glutathione.

13. The method according to claim 1 or 2, wherein the dried biodegradable PEG-based insoluble hydrogel is solvated with 5-10ml NMP per g of dried biodegradable PEG-based insoluble hydrogel using a closed container and irradiated with gamma rays at a dose of 25 kGy.

14. A sterilized biodegradable PEG-based insoluble hydrogel obtainable by the method of claim 1.

15. Use of the hydrogel of claim 14 in tissue engineering, skin augmentation, intraocular devices, medical implants, surgical sealants and sponges, hemostats, sustained release drug delivery systems, medical imaging agents and prodrug carriers.