Patent Application
20220241194
The present invention relates to cross-linkable hydrogels and their use as inks in direct ink writing (DIW) or their use as injectable vehicles, which hydrogels exhibit shear-thinning as well as self-healing.
A hydrogel is a network of polymer chains that are generally hydrophilic in which water forms the phase in which the polymer chains are dispersed. Examples include plant or animal biopolymers such as collagen, alginate, hyaluronic acid or synthetic polymers such as poly(ethylene glycol) diacrylate (PEGDA).
Hydrogels may be useful in several applications and in particular can be useful in biological, biomedical or pharmaceutical applications where they may serve as either scaffolds for tissue engineering, wound repair materials, vehicles in injectable sustained release formulations or as biological models.
In said applications, the hydrogels are generally brought into a certain shape via several techniques, such as for example additive manufacturing (AM), which enables freeform and accurate deposition of the hydrogels, enabling on-demand fabrication of objects.
Alternatively, cells- or drug-containing hydrogels may be injected directly into a corporeal site of medical application to form, for example, a localized depot body through which cells can migrate out- or towards it, or a drug is controllably released.
Efficient fabrication of biomaterial objects via AM requires suitable inks. Such inks need to satisfy specific physical constraints, defined by the AM technology, related to the material viscosity, flowability, self-healing rate, and gelation kinetics. The target application often introduces additional design parameters. For example, in tissue engineering the ink should be compatible with living cells while in drug delivery the ink should enable the controlled release of molecular therapeutics. To date, a range of inks tailored to a specific AM technology and/or application exist, enabling to provide constructs that can be used in tissue repair, for ex vivo drug screening and disease modeling, or for the design of patient-specific drug delivery systems (DDS).
Direct ink writing (DIW) is one of the main AM technologies used in particular for bio-fabrication applications. DIW operates by extruding a material ink via piston-, pneumatic-, or screw-driven robotic dispensing in defined locations to fabricate the final 3D construct. In general, inks suitable for DIW must demonstrate shear-induced flow during extrusion (i.e. shear-thinning) and rapid material reformation for shape retention following deposition (i.e. self-healing). On the other hand, shear-induced flow and shape retention immediately after deposition are also properties that are desirable in injectable formulations that may be used for sustained drug release and delivery.
While effectively bringing the hydrogels into a certain form is important, long-term shape retention and/or structural integrity of the hydrogel structure in the environment of intended use such as aqueous environments and/or biological environments is equally important.
US2017/0319506 A1 discloses shear thinning and self-healing polymer nanoparticle (PNP) gels comprising one or more gel-forming polymer and a nanoparticle. The gels can be used in biomedical application such as drug delivery systems for in vitro or in vivo study.
However, when the gels of US2017/0319506 A1 are exposed to an aqueous environment such as water or buffer solutions, they quickly erode and disintegrate, thereby limiting their use in aqueous or biological environments and in particular in long-term drug screening and disease modeling or in depot formulations.
It is therefore desirable to provide inks which are suitable for DIW in the sense that they should exhibit shear-thinning, self-healing and which moreover allow for the manufacture of constructs having extended structural stability in aqueous environments and thus may render possible their use both in long-term drug screening and disease modeling or in extended release formulations, in in vitro and in vivo applications.
The present applications therefore provides a cross-linkable hydrogel composition that in addition to exhibiting shear thinning and self-healing also exhibits dimensional stability in aqueous and/or biological environments. The hydrogels according to the present invention are furthermore unexpectedly robust in the sense that they can be accommodate a variety of cross-linkable hydrogel precursors comprised in the cross-linkable hydrogel composition of the present invention.
It is an object of the present invention to provide a cross-linkable hydrogel composition comprising an aqueous base carrier composition and a cross-linkable hydrogel precursor, wherein the aqueous base carrier composition comprises one or more polymers and one or more nanoparticles, wherein the one or more polymers are selectively adsorbed to the one or more nanoparticles, and wherein the one or more polymers and the one or more nanoparticles form a transient shear-thinning and self-healing hydrogel and wherein the one or more polymers and the one or more nanoparticles are each comprised in the cross-linkable hydrogel composition in a concentration at which neither the one or more polymers nor the nanoparticles, taken alone, form a hydrogel, characterized in that the cross-linkable hydrogel precursor is not selectively adsorbed to the one or more nanoparticles and in that the cross-linkable hydrogel precursor and the one or more nanoparticles do not form a transient shear-thinning and self-healing hydrogel and further in that the cross-linkable hydrogel precursor is comprised in the cross-linkable hydrogel composition in a concentration at which the cross-linkable hydrogel precursor, taken alone, does not form a hydrogel.
In the context of the present invention, the term “gel” and “hydrogel” relates to materials in which the storage modulus (G′) is greater than the loss modulus (G″) at a frequency of 10 rad/s, as measured by oscillatory shear rheometry in the linear viscoelastic regime.
In a preferred embodiment of the cross-linkable hydrogel compositions according to the present invention, the one or more nanoparticles may comprise or consist of an inorganic or organic material. Exemplary materials are inorganic materials such as silica, titania, or organic materials such as synthetic polymers.
In a preferred embodiment of the cross-linkable hydrogel compositions according to the present invention, the one or more nanoparticles may comprise or consist of one or more block copolymers, in particular amphiphilic diblock copolymers, for example amphiphilic diblock copolymers having a polyether block such as PEG-b-PLA, PEG-b-PGA PEG-b-PCL, PEG-b-PLGA, PEG-b-PE PEG-b-PS where PEG is polyethylene glycol, PLA is polylactic acid, PGA is polyglycolic acid, PLGA is polylactic-co-polyglycolic acid, PCL is polycaprolactone, PE is polyethylene and PS is polystyrene. In an alternative embodiment, the one or more nanoparticles comprise, or consist of, PEGylated inorganic nanoparticles such as PEGylated iron oxide nanoparticles or PEGylated silica nanoparticles.
The one or more nanoparticles comprising or consisting of one or more amphiphilic diblock copolymers may be obtained by first dissolving the copolymers in a suitable organic solvent and then adding the solution dropwise into an aqueous or water phase under shear such as to precipitate the solved copolymer and form the nanoparticles.
In a preferred embodiment of the cross-linkable hydrogel compositions according to the present invention, the one or more nanoparticles may comprise or consist of PEG-b-PLA.
In a preferred embodiment of the cross-linkable hydrogel compositions according to the present invention, the cross-linkable hydrogel precursor and in particular in the case where the cross-linkable hydrogel precursor is a semisynthetic homo- or heteropolysaccharide, the polysaccharide may be substituted with acrylate or methacrylate moieties. Such a polysaccharide may be methacrylated hyaluronic acid or acrylated hyaluronic acid.
The degree of substitution of the polysaccharide substituted with acrylate or methacrylate moieties may be of from 0.1 to about 0.5, preferably of from 0.25 to 0.45 as determined by 1H-NMR in D2O from integration of the vinyl group normalized to the polysaccharide backbone. It has been found that too high a degree of substation of the polysaccharide negatively affect shelf life of the cross-linkable hydrogel compositions according to the present invention because of phase separation between the cross-linkable hydrogel precursor and the base carrier composition.
In a preferred embodiment of the present invention, the polyethylene glycol block in the diblock copolymer may be from 1 000 Da to 25 000 Da, preferably of from 1 000 Da to 5 000 Da and/or the polyhydroxy alkanoate block, and in particular the polylactic acid block, may be from 5 000 Da to 100 000 Da and preferably from 12 000 Da to 20 000 Da.
The shear-thinning and self-healing networks of the base carrier composition are formed by the appropriately paired nanoparticles and polymers, which selectively adsorb to the nanoparticles to form non-covalent cross-links that in turn lead to gel formation. Owing to the dynamic and transient nature of these cross-links, the base carrier composition exhibits dramatic shear-thinning and rapid self-healing.
The polymers and nanoparticles that form the shear-thinning and self-healing hydrogels of the base carrier composition each independently do not form a gel alone or are not used at a concentration where the polymer alone or nanoparticle alone form a gel. Only when the nanoparticle and polymer are combined does gel formation occur.
Thus, in the cross-linkable hydrogels according to the present invention, the loss modulus (G″) for a solution of the polymer of the base carrier composition and the loss modulus (G″) of a solution of the one or more nanoparticles of the base carrier composition are each greater than their respective storage moduli (G′) at a frequency of 10 rad/s as measured by oscillatory shear rheometry in the linear viscoelastic regime; and wherein the one or more nanoparticles and the polymer of the base carrier composition form a hydrogel when combined. The storage modulus (G′) for the resulting hydrogel is greater than the loss modulus (G″) at a frequency of 10 rad/s as measured by oscillatory shear rheometry in the linear viscoelastic regime when combined.
In a preferred embodiment of the cross-linkable hydrogel compositions according to the present invention, the one or more nanoparticles have particle size Dh of between 20 nm and 100 nm, preferably between 45 nm and 65 or between 45 nm and 75 nm and/or the nanoparticles are present in an amount of between 1 and 25 wt % based on the weight of the cross-linkable hydrogel composition, and preferably between 10 and 20 wt % or between 5 and 20 wt % or between 2 and 20 wt %, based on the weight of the cross-linkable hydrogel composition. The Dh can be determined by dynamic light scattering at a scattering angle of 173° at 25° C.
In a preferred embodiment of the cross-linkable hydrogel compositions according to the present invention, the cross-linkable hydrogel precursor is selected from the group consisting of synthetic or semisynthetic polymers, preferably bearing acrylate or methacrylate moieties, and more preferably having a degree of substitution of less than 0.5.
In a preferred embodiment of the cross-linkable hydrogel compositions according to the present invention, the one or more polymer of the aqueous base carrier composition is selected from the group consisting of semisynthetic polysaccharides, in particular from the group consisting of semisynthetic cellulose ethers or mixtures thereof such as methyl cellulose, ethyl cellulose, ethyl methyl cellulose, hydroxypropylmethyl cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose and most preferably is hydroxypropylmethyl cellulose or hydroxyethyl cellulose, or mixtures thereof.
In a preferred embodiment of the cross-linkable hydrogel compositions according to the present invention, the one or more polymer of the aqueous base carrier composition, in particular in the case where the one or more polymer is a semisynthetic cellulose ether, has a molecular weight of from 100 kDa to 850 kDa, or from 10 kDa to 850 kDa, preferably of from 250 to 750 kDa, or from 50 kDa to 750 kDa.
In a preferred embodiment of the cross-linkable hydrogel compositions according to the present invention, the cross-linkable hydrogel composition further comprises one or more active pharmaceutical ingredients, in particular wherein the one or more active pharmaceutical ingredients are confined to the one or more nanoparticles of the base carrier composition. In the case where one or more active pharmaceutical ingredients are confined to the one or more nanoparticles, the one or more nanoparticles comprise, or consist of an amphiphilic diblock copolymer such as PEG-b-PLA, PEG-b-PGA PEG-b-PCL, PEG-b-PLGA, PEG-b-PE PEG-b-PS where PEG is polyethylene glycol, PLA is polylactic acid, PGA is polyglycolic acid, PLGA is polylactic-co-polyglycolic acid, PCL is polycaprolactone, PE is polyethylene and PS is polystyrene.
In a preferred embodiment of the cross-linkable hydrogel compositions according to the present invention, the cross-linkable hydrogel compositions further comprises one or more cells, preferably microorganism cells. The cells may be from yeasts, bacteria, plants or animals.
In a preferred embodiment of the cross-linkable hydrogel compositions according to the present invention, the cross-linkable hydrogel precursor is cross-linkable by radiation, change of pH, change of temperature or by contact with a crosslinking agent such as for example an ionic crosslinking agent.
The radiation wavelength needed for cross-linking the cross-linkable hydrogel compositions according to the present invention will depend on the type of cross-linkable hydrogel precursor used, and may for example be IR, visible or UV radiation. In the case where the cross-linkable hydrogel precursor is a synthetic or semisynthetic polymer bearing acrylate or methacrylate moieties, a suitable wavelength may be 405 nm.
The crosslinking agent needed for cross-linking the cross-linkable hydrogel compositions according to the present invention will depend on the type of cross-linkable hydrogel precursor used, and may for example be an ionic crosslinking agent such as metal ions. For instance, a suitable ionic crosslinking agent in the case where alginate is used as the cross-linkable hydrogel precursor may for example be divalent metal cations such as calcium or barium cations.
In a preferred embodiment of the cross-linkable hydrogel compositions according to the present invention, the cross-linkable hydrogel composition further comprises a photoinitiator capable of cross-linking the cross-linkable hydrogel precursor upon exposure to a cross-linking radiation, in particular when the cross-linkable hydrogel precursor is cross-linkable by radiation, such as when the cross-linkable hydrogel precursor is a synthetic or semisynthetic polymer bearing acrylate or methacrylate moieties, A suitable photoinitiator capable of cross-linking the cross-linkable hydrogel precursor upon exposure to a cross-linking radiation is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), which may be comprised in the cross-linkable hydrogel composition according to the present invention in an amount of about 0.05 to 1 wt %, based on the weight of the cross-linkable hydrogel composition.
In a preferred embodiment of the cross-linkable hydrogel compositions according to the present invention, the cross-linkable hydrogel precursor is chosen from animal or plant homo- or heteropolysaccharides, such as alginate, and derivatives thereof, in particular methacrylated derivatives thereof such as methacrylated hyaluronic acid.
In a preferred embodiment of the cross-linkable hydrogel compositions according to the present invention, the cross-linkable hydrogel precursor is chosen from animal or plant homo- or heteropolysaccharides substituted with acrylate or methacrylate moieties, and which may have a degree of substitution of from 0.1 to about 0.5, preferably of from 0.25 to 0.45 as determined by 1H-NMR in D2O from integration of the vinyl group normalized to the polysaccharide backbone.
In a preferred embodiment of the cross-linkable hydrogel compositions according to the present invention, the cross-linkable hydrogel precursor is a protein such as collagen. Alternatively, the cross-linkable hydrogel precursor may be a protein such as laminin, fibronectin, or any natural or semisynthetic protein. Collagen can be cross-linked, as is known in the art, by controlling the pH such as to be between 6.5 to 8.5; preferably between 7.6 to 8.0 in an aqueous buffer of adjusted ionic strength. Suitable buffers can be based on PBS or HEPEs in addition to some salts such as for example NaCl.
In a preferred embodiment of the cross-linkable hydrogel compositions according to the present invention, the cross-linkable hydrogel precursor is a synthetic polymer, in particular a methacrylated or acrylated synthetic polymer such as a methacrylated or acrylated poly(propylene glycol), poly(ethylene glycol) diacrylate or poly(ethylene propylene glycol), diacrylate.
It is further an object of the present invention to provide a use of a cross-linkable hydrogel composition according to the above as an ink for direct ink writing. The cross-linkable hydrogel composition exhibits shear-thinning and self-healing which allows for convenient extrusion through the nozzle of a direct ink writing apparatus and affords the self-support needed in a green state before cross-linking of the cross-linkable hydrogel precursor is induced, which cross-linking provides for stability in aqueous or biological environments.
It is further an object of the present invention to provide a use of a cross-linkable hydrogel composition according to the above as a vehicle for an injectable pharmaceutical depot formulation.
The cross-linkable hydrogel composition exhibits shear-thinning and self-healing which allows for convenient injection, for example via a syringe, into an injection site in a tissue before cross-linking of the cross-linkable hydrogel precursor is induced, which cross-linking provides for stability at the site of injection. In the case where the cross-linkable hydrogel composition includes an active pharmaceutical ingredient, the cross-linked cross-linkable hydrogel composition exhibits a controlled release of the active pharmaceutical ingredient, in particular when the active pharmaceutical ingredient is bound to the one or more nanoparticles. Because the nanoparticles are released more slowly from a depot of cross-linked cross-linkable hydrogel composition of the present invention when compared to a depot consisting of solely the base carrier composition, the release of the active pharmaceutical ingredient can be more accurately controlled.
It is further an object of the present invention to provide an object, obtained by cross-linking a green object of cross-linkable hydrogel composition according to the above.
The green object may be obtained in general by subjecting the cross-linkable hydrogel composition to shear such as to fluidize it and depositing the fluidized cross-linkable hydrogel composition, for example by direct ink writing the cross-linkable hydrogel composition or by injecting the cross-linkable hydrogel composition, prior to cross-linking the cross-linkable hydrogel composition.
It is further an object of the present invention to provide a process of manufacturing a object comprising the steps of
It is further an object of the present invention to provide a use for an aqueous base carrier composition in a DIW ink composition further comprising a cross-linkable hydrogel precursor, the aqueous base carrier composition comprising one or more hydrogel-forming polymers and one or more nanoparticles, wherein the one or more hydrogel-forming polymers are selectively adsorbed to the one or more nanoparticles, and wherein the one or more polymers and the one or more nanoparticles form a shear-thinning and self-healing hydrogel, and wherein the one or more hydrogel-forming polymers and the one or more nanoparticles are each comprised in the aqueous base carrier composition in a concentration at which neither the one or more polymers nor the nanoparticles, taken alone, form a hydrogel, characterized in that the cross-linkable hydrogel precursor is not selectively adsorbed to the one or more nanoparticles and in that the cross-linkable hydrogel precursor and the one or more nanoparticles do not form a shear-thinning and self-healing hydrogel and preferably in that the cross-linkable hydrogel precursor is comprised in the DIW ink composition in a concentration at which the cross-linkable hydrogel precursor, taken alone, does not form a hydrogel.
Further embodiments of the invention are laid down in the dependent claims.
Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,
Materials
Hydroxypropylmethylcellulose (HPMC; Mn˜700 kDa, Ref. H3785), methacrylic anhydride (MA; Ref 276685), 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (Hepes; 99%, Ref H3375), Oil Red O (OR; Ref 00625), calcium chloride (CaCl2; Ref 21074), anti-mouse IgG-Atto 594 (IgG-Atto 594; Ref 76085), 2,4,6-trimethylbenzoyl chloride (Ref 682519), dimethyl phenylphosphonite (Ref 149470), lithium bromide (Ref 213225), DAPI (Ref D9542) and 2-butanone (Ref 360473) were purchased from Sigma-Aldrich (Buchs, Switzerland). PEG5k-b-PLA16k (AK054) and PEG5k-b-PLA14k (AK049) were purchased from Akina (West Lafayette, Ind., USA). Sodium alginate (Alg; Grade I-1G-80) was purchased from Kimica International (Tokyo, Japan); poly(ethylene glycol) diacrylate (PEGDA; Mw ˜10 kDa, Ref ACRL-PEG-ACRL-10K) was purchased from Laysan Bio Inc. (Arab, Ala., USA). Sodium hyaluronate (HA; Mw ˜700 kDa, Ref HA700K) was purchased from Lifecore (Chaska, Minn., USA). Collagen (Col; 10 mg/ml, Ref 5133-20ML) was purchased from Advanced BioMatrix (San Diego, Calif., USA). Acetone (Ref 20066.296) and acetonitrile (Ref 20071.294) were purchased from VWR Chemicals. Low glucose DMEM (Ref 22320-022), Pen Strep (PS, Ref 15140-122), FGF (Ref PHG0026), and fetal bovine serum (FBS; Ref 10270106), LIVE/DEAD Viability/Cytotoxicity kit (Ref L3224), AlexaFluor 488 Phalloidin (Ref A12379) were purchased from ThermoFisher.
Preparation of PEG-b-PLA Nanoparticles
The PEG-b-PLA nanoparticles were obtained by dissolving 140 mg PEG-b-PLA in 2 ml of acetone, from which solution the PEG-b-PLA nanoparticles were nanoprecipitated by dropwise addition (15 μL every 3 s) of said solution into 10 ml Milli-Q water under a high stir rate (650 rpm). After overnight evaporation of acetone, the thus obtained PEG-b-PLA nanoparticles were filtered by ultracentrifugation and re-suspended to a final concentration of 20 wt % in aqueous buffer (20 mM HEPES and 275 mM mannitol) or [for UNI, UNI-PEGDA, UNI-MeHA, and UNI-Alg] or to a final concentration of 25 wt % [for UNI-Col)] in Milli-Q water.
The size of the PEG-b-PLA nanoparticles (Dh˜55 nm) and dispersity of the PEG-b-PLA nanoparticles (PDI ˜0.1) were characterized by dynamic light scattering on a Malvern ZetaSizer Nano ZS at a scattering angle of 173° and at 25° C.).
Preparation of Base Carrier Composition Using PEG-b-PLA Nanoparticles and HPMC (UNI-15)
An aqueous base carrier composition of HPMC and PEG-b-PLA nanoparticles was prepared by first dissolving HPMC in buffer solution (20 mM HEPES and 150 mM D-mannitol, pH=7.4) at 1 wt % and allowed to equilibrate overnight (25° C.). Thereafter, the HPMC solution was combined with the 20 wt % nanoparticles in aqueous buffer (20 mM HEPES and 275 mM mannitol) to yield a base carrier composition of 15 wt % PEG-b-PLA nanoparticles and 1 wt % HPMC.
A composition was prepared by first dissolving 1 wt % HPMC in an aqueous buffer (20 mM HEPES and 150 mM D-mannitol, pH=7.4) together with 1 wt % alginate and allowed to equilibrate overnight (25° C.). This composition comprising 1 wt % alginate and HPMC, respectively, was then combined and mixed with the above suspension of 20 wt % PEG-b-PLA nanoparticles in aqueous buffer (20 mM HEPES and 275 mM mannitol) such as to provide for the cross-linkable hydrogel composition according to the present invention comprising 1 wt % alginate, 15 wt % PEG-b-PLA nanoparticles and 1 wt % HPMC.
An aqueous base carrier composition according to the present invention comprising 1 wt % methacrylate hyaluronic acid, 15 wt % PEG-b-PLA nanoparticles and 1 wt % HPMC was obtained in the same manner as above, with the exception that 1 wt % methacrylate hyaluronic acid was dissolved together with 1 wt % HPMC and 0.1 wt % lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as photoinitiator in an aqueous buffer (20 mM HEPES and 150 mM D-mannitol, pH=7.4).
The Methacrylate Hyaluronic had a Degree of Substitution of about 0.4.
An aqueous base carrier composition according to the present invention comprising 1 wt % poly(ethylene glycol) diacrylate, 15 wt % PEG-b-PLA nanoparticles and 5 wt % HPMC was obtained in the same manner as above, with the exception that 1 wt % poly(ethylene glycol) diacrylate was dissolved together with 5 wt % HPMC and 0.1 wt % lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as photoinitiator in an aqueous buffer (20 mM HEPES and 150 mM D-mannitol, pH=7.4).
A composition was prepared by first dissolving 1 wt % HPMC in Milli-Q water and allowed to equilibrate overnight (4° C.). This composition comprising 1 wt % HPMC was then combined and mixed with the above suspension of 25 wt % PEG-b-PLA nanoparticles in Milli-Q water such as to provide for the aqueous base carrier composition of the cross-linkable hydrogel composition according to the present invention comprising 1 wt % alginate, 15 wt % PEG-b-PLA nanoparticles and 1 wt % HPMC.
Collagen was combined and mixed with the aqueous base carrier composition and until a concentration of 0.2 wt % collagen, 1 wt % HPMC and 15 wt % PEG-b-PLA nanoparticles reached.
A composition was prepared by first dissolving 1 wt % HEC in Milli-Q water and allowed to equilibrate overnight (4° C.). This composition comprising 1 wt % HEC was then combined and mixed a suspension of 25 wt % silica nanoparticles in Milli-Q water such as to provide for the aqueous base carrier composition of the cross-linkable hydrogel composition according to the present invention comprising 3 wt % methacrylate dextran, 15 wt % silica nanoparticles and 1 wt % HEC.
The methacrylate dextran had a degree of substitution of about 0.7.
While the composition exhibited desirable shear thinning and self-healing, the shelf life was limited in the sense that the composition would phase-separated within 24 hrs whereas with the other exemplary compositions no phase separation could be observed even after weeks or months.
Cross-Linking of the Cross-Linkable Hydrogel Compositions
The alginate cross-linkable hydrogel composition of Example 1 was cross-linked by immersing the scaffold in 100 mM CaCl2 solution for 30 min.
The PEGDA and MeHA cross-linkable hydrogel compositions of Example 2 and 3 were cross-linked with visible light (k=405 nm, at I=15 mW cm−2) for 5 min.
The collagen cross-linkable hydrogel composition of Example 4 was cross-linked by placing in an incubator (37° C., 5% CO2) for 120 min.
Results
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Thus, the cross-linkable hydrogel compositions according to the present invention exhibit extended stability and structural integrity in aqueous environments.
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| Number | Date | Country | Kind |
|---|---|---|---|
| 19181458.1 | Jun 2019 | EP | regional |
This application is a national phase entry under 35 U.S.C. § 371 of International Patent Application PCT/EP2020/067187, filed Jun. 19, 2020, designating the United States of America and published in English as International Patent Publication WO 2020/254626 on Dec. 24, 2020, which claims the benefit under Article 8 of the Patent Cooperation Treaty to European Patent Application Serial No. 19181458.1, filed Jun. 20, 2019, the entireties of which are hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/067187 | 6/19/2020 | WO | 00 |
