Hydrogels for use as vitreous body substitute

Abstract

Disclosed are hydrogels and pre-gel products for use an artificial vitreous body. Methods and uses of such hydrogels as drug delivery tools and / or for the treatment of diseases, and devices for administration of said hydrogels and pre-gel products are disclosed as well.

Description

[0001]Hydrogels for use as vitreous body substitute Disclosed are hydrogels and pre-gel products for use an artificial vitreous body. Methods and uses of such hydrogels as drug delivery tools and / or for the treatment of diseases, and devices for administration of said hydrogels and pre-gel products are disclosed as well. the Disclosure The vitreous body, the gel-like substance that fills the vitreous chamber of the eye, is a complex structure, and developing a synthetic replacement that mimics its functions has proven chal- lenging. In case of diseases or traumata the natural vitreous humor needs to be partially or fully substituted by artificial replacements. Prior art vitreous body substitutes such as gases or silicone oil, which are used, for example, after a retinal detachment, show serious limitations. Silicone oil is a chemically inert material. However, oil bubbles (emulsification) occur in the patient's eye after just a few months. The sur- gical handling of the oil is time-consuming and since a fat-soluble environment is created, the behavior of drugs that are injected into the eye is significantly changed depending on their chemical properties. Gas mixtures, which may be used, are absorbed within a few days to weeks and, thus, lose their effects after a short time. Further, both materials, gas and silicone oil, limit the patient's vision after the operation. Also, the tamponade function is not optimal: gas and light silicone oil work with buoyancy. Therefore, in complicated cases, reattachment of the retina cannot be guaranteed for the whole area and patients are forced to assume uncomfortable head positions to direct the buoyancy of the sub- stitutes to the site of retinal detachment. Recently hydrogels with fast-transition from sol- to gel-state were examined as artificial vitreous body. Although some hydrogels can be introduced into the body via injection of a polymer solu- tion followed by in situ polymer crosslinking, conventional hydrogels often swell, which compro- mises their morphological and mechanical compatibility in vivo. Further, and most importantly, existing fast-forming hydrogels turned out to be cytotoxic and were therefore not suitable for medical uses as artificial vitreous bodies. Hayashi et al., 2017 (Nature Biomedical Engineering, March 9, 2017, Vol.1) refer to hydrogels which can be used as artificial vitreous bodies. Hayashi et al. claim to have found non-toxic hy- drogels for that purpose. However, ARPE-19-cell-assays (a specific cell line derived from the human retinal pigment epithelium (RPE)) conducted in the present disclosure, revealed a cell- April 4, 2025 1 / 43 cytotoxicity of the hydrogels used in Hayashi et al., 2017. Thus, need exists to develop novel artificial vitreous body substitutes which overcome the prob- lems of the prior art. Brief Summary of the Disclosure In a first aspect the disclosure relates to a product comprising a first and a second component as a combined preparation for simultaneous or sequential use as an artificial vitreous body, a. wherein the first component comprises tetra-PEG-cyclooctyne and tetra-PEG- azide, wherein tetra-PEG-cyclooctyne is present in molar excess as compared to tetra-PEG-azide, and b. wherein the second component comprises tetra-PEG-azide and tetra-PEG-cy- clooctyne wherein tetra-PEG-azide is present in molar excess as compared to tetra-PEG-cyclooctyne. Wherein “simultaneous use” means that the two components may be administered at the same time into the eye. Wherein “simultaneous use” also encompasses that the two components may be mixed shortly before injection into the eye, that is in one embodiment less than 20 minutes before, in another embodiment less than 15 minutes before, in another embodiment less than 10 minutes before. In yet another embodiment “simultaneous use” also encompasses that the two components may be mixed during injection into the eye. Wherein “sequential use” means that the two components may be administered shortly after each other into the eye, with a time gap between the two injections in one embodiment of less than 20 minutes apart, in another embodiment of less than 15 minutes apart, in yet another em- bodiment of less than 10 minutes apart, in yet another embodiment of less than 5 minutes apart. In a second aspect the disclosure relates to a method for producing a product comprising a combined preparation for simultaneous or sequential use as an artificial vitreous body compris- ing the steps of: a. Provision of a first component comprising tetra-PEG-cyclooctyne and tetra-PEG- azide, wherein tetra-PEG- cyclooctyne is in molar excess as compared to tetra- PEG-azide, and b. Provision of a second component comprising tetra-PEG-azide and tetra-PEG- cyclooctyne wherein tetra-PEG-azide is present in molar excess as compared to April 4, 2025 2 / 43 tetra-PEG-cyclooctyne, c. Storing the first and the second component separately for at least 1 hour at 20°C; d. Optionally, lyophilizing the first and / or the second component. In a third aspect the disclosure relates to a method for obtaining a hydrogel for use as an artifi- cial vitreous body comprising the steps a - c, optionally a – d, of before-mentioned method, comprising further the steps of: e. Optionally, reconstituting the first and / or the second lyophilized component, f. Mixing the first and the second component in a ratio (vol.-%) from 3:1 to 1:3, pref- erably in a ratio (vol.-%) of about 1:1, g. thereby obtaining the hydrogel in sol-state. In a fourth aspect the disclosure relates to a product produced by the method according to the second aspect and / or a hydrogel produced by the method according to the third aspect. In a fifth aspect the disclosure relates to a product and / or hydrogel, comprising further an active agent for use as a drug-release system in the eye. In a sixth aspect the disclosure relates to a product and / or hydrogel, for use in the treatment of a condition or disease selected from retinal detachment, diabetic retinopathy, macular degener- ation, traumatic eye injuries, vitreous haemorrhage, and infections in the eye interior (vitreous). In a seventh aspect the disclosure relates to device for intravitreal injection into the eye, com- prising a first chamber with a first component and a second chamber with a second component, a. wherein the first component in the first chamber comprises tetra-PEG-cy- clooctyne and tetra-PEG-azide, wherein tetra-PEG-cyclooctyne is present in mo- lar excess as compared to tetra-PEG-azide, and b. wherein the second component in the second chamber comprises tetra-PEG-az- ide and tetra-PEG-cyclooctyne wherein tetra-PEG-azide is present in molar ex- cess as compared to tetra-PEG-cyclooctyne. The first and the second component may be stored separately until they are mixed to produce the hydrogel. April 4, 2025 3 / 43 Brief Description of the Figures Figure 1 – Depiction of the preparation of the first and second component (component A and B), and the hydrogel from tetra-PEG-monomers with azide, respectively, cyclooctyne active groups. The first and second component differ from each other in that tetra-PEG-cyclooctyne is present in molar excess as compared to tetra-PEG-azide in the first component, and tetra-PEG- azide is present in molar excess as compared to tetra-PEG-cyclooctyne in the second compo- nent. Both components are stored overnight at 20°C in order to allow full polymerization of each component. The hydrogel may then be formed outside the eye shortly (< 15 minutes) prior to injection, or during or after injection into the eye just by mixing the two components together in a ratio (vol.%) from 3:1 to 1:3, preferably about 1:1. Figure 2 - Gelation of the gel after mixing the pre-gels reaches the gelation point after around 10 minutes. Figure 3 - Both, forward light scattering (A) and turbidity (B), are lower in the presented hydro- gel compared to juvenile, porcine vitreous bodies. Figure 4 - After the aging procedure, the gel still exhibits optimal viscoelastic properties (B) and has very low straylight (A). Figure 5 - The monomers of the present disclosure showed no cytotoxicity even in high concen- trations. Figure 6. - The gel showed no cytotoxicity for the needed concentrations. Figure 7 - Microscopic images of the ARPE 19 cells after incubation with Presto Blue®. The cells were treated with the gel extract 10 g / L (a), Triton X 1% as negative control (b) and the complete medium as positive control (c). Images were recorded with a Zeiss Primovert®inverse microscope. Figure 8 – Reaction to create functional oligomers only takes 2-4 hours compared to Hayashi et al., 2017 with >12 hours. Figure 9 - Frequency Sweep after 3 months of incubation in balanced salt solution. Figure 10 - Optical impact of liposomes and PLGA-nanoparticles incorporated into the hydrogel platform. Figure 10 A) optical impact of liposomes and PLGA-nanoparticles incorporated into the hydrogel platform; Figure 10 B) Liposomal-nanoparticles; Figure 10 C) PLGA-nanoparticles; Fig- ure 10 D) modulation transfer function. April 4, 2025 4 / 43 Figure 11 – Corresponding characteristics of PLGA-nanoparticles und liposomes for Figure 10. Figure 12 – Storage and loss modulus for the highest tested concentration of liposomes and PLGA-nanoparticles (0.5 mg / ml) are comparable to the hydrogel platform without any additions. Figure 13 - Gel network does not change due to the addition of liposomes (right side, circled). Figure 14 - PEG2000 – in-vivo biocompatibility in landrace pigs. Figure 15 - Electroretinographically results from baseline measurements (B) as well as 2 and 4 weeks after injection of 40 mg PEG 2000 (n=3). April 4, 2025 5 / 43 Detailed Description of the Disclosure Product The hydrogel suitable as an artificial vitreous body of the present disclosure is composed of two components (also called “pre-gel” throughout this disclosure), wherein the first component com- prises tetra-PEG-cyclooctyne and tetra-PEG-azide, wherein tetra-PEG-cyclooctyne is present in molar excess as compared to tetra-PEG-azide, and wherein the second component comprises tetra-PEG-azide and tetra-PEG-cyclooctyne wherein tetra-PEG-azide is present in molar excess as compared to tetra-PEG-cyclooctyne. In one embodiment the first component comprises tetra-PEG-cyclooctyne in a molar ratio r of at least 0.51, of at least 0.55, of at least 0.60, of at least 0.65, of at least 0.70, or of at least 0.75. In another embodiment the first component comprises tetra-PEG-cyclooctyne in a molar ratio r of up to 0.95, of up to 0.90, of up to 0.85, or of up to 0.80. In yet another embodiment the first com- ponent comprises tetra-PEG-cyclooctyne in a molar ratio r of from 0.51 to 0.95, of from 0.55 to 0.90, of from 0.60 to 0.85, or of from 0.65 to 0.80. In all of the before-mentioned embodiments the first component comprises also tetra-PEG-azide in a molar ratio r of 1-r, wherein r = [tetra- PEG-cyclooctyne] in mol / ([tetra-PEG-cyclooctyne] in mol + [tetra-PEG-azide] in mol). In one embodiment the second component comprises tetra-PEG-azide in a molar ratio s of at least 0.51, of at least 0.55, of at least 0.60, of at least 0.65, of at least 0.70, or of at least 0.75. In another embodiment the second component comprises tetra-PEG-azide in a molar ratio s of up to 0.95, of up to 0.90, of up to 0.85, or of up to 0.80. In yet another embodiment the second component comprises tetra-PEG-azide in a molar ratio s of from 0.51 to 0.95, of from 0.55 to 0.90, of from 0.60 to 0.85, or of from 0.65 to 0.80. In all of the before-mentioned embodiments the second component comprises also tetra-PEG- cyclooctyne in a molar ratio s of 1-s, wherein s = [tetra-PEG-azide] in mol / ([tetra-PEG-azide] in mol + [tetra-PEG- cyclooctyne] in mol). Thus, in one embodiment the disclosure relates to a product comprising the first and the second component as a combined preparation for simultaneous or sequential use as an artificial vitre- ous body. It has been found that these two components are non-cytotoxic and form a non-cyto- toxic hydrogel upon mixing of each other, and, therefore, can be used as an artificial vitreous body. This differentiates the present components from prior art hydrogel-forming components which may react to proteins and other biological substances in the target tissue, thereby being toxic and / or leading to unwanted side-reactions. In contrast, the components of the present dis- closure and the hydrogel comprise functional groups which do not occur in the body and do not cross react with proteins of the retina. April 4, 2025 6 / 43 The two components may be stored in solution and / or as lyophilizate for at least 1 hour, at least 6 hours, at least 12 hours, at least 24 hours, at least 2 days, at least 5 days, at least 7 days, at least 2 weeks, or at least 1 months. In one embodiment the two components may be stored in solution and / or as lyophilizate for up to 2 years, up to 1.5 years, up to 1 year, or up to 6 months. The storage may be in one embodiment at room temperature (20°C), in another embodiment at 4°C, in yet another embodiment at -20°C, or in yet another embodiment at -80°C. In one embodiment the first and / or second component may be in a solution further comprising a sterile buffered saline at a pH from 6.5 to 8.5, at a pH from 6.8 to 7.8, at a pH from 7.0 to 7.4, or at a pH of about 7.4 (i.e. physiological pH). In one embodiment the first and / or second compo- nent may have a pH of 7.4, (T = 36-37,4°C), and an osmolarity of about 300 mOsm / L. In one embodiment the first and / or second component may be lyophilized. In one embodiment the first and / or second component may therefore further comprise a freezing agent, such as a cryoprotectant selected from sucrose, lactose, palatinose, trehalose, cellobiose, mannitol, glu- cose, sorbitol and / or amino acids, a buffer, a salt, a polymer such as polyvinylpyrrolidone (PVP), polyvinyl alcohole (PVA) and / or hydroxyethyl starch (HES), and / or surfactants such as polysorb- ate. The hydrogel of the present disclosure also shows a good long-term stability (i.e. no changes in viscoelastic properties and forward light scattering) for at least 7 days, at least 2 weeks, or at least 1 month. In one embodiment the hydrogel of the present disclosure is stable for up to 5 years, up to 4 years, up to 3 years, up to 2 years, up to 1.5 years, up to 1 year, or up to 6 months. The first and second component, as well as the hydrogel in its sol-state (within the first 10-20 minutes, preferably within the first 15 minutes after mixing the first and second component) al- lows the injection in the smallest gauge used in in retinal surgery, (also only in rare, very elec- tive cases), which is 27-G. The first and second component can be easily injected through 30G- cannulas, most likely even smaller ones without any problem. This is in strong contrast to other viscoelastic ophthalmic devices using during surgery (only intraoperatively) and pre-linked hy- drogels (e.g. Schulz et al, TVST, attached). The first and second component require <10 New- ton of force to be injected through a 23G-cannula, while a prelinked alginate gel previously has to be forced through using more than 60-70 N. Thus, in one embodiment the first and second component of the present disclosure, as well as the hydrogel in its sol-state, is characterized by being injectable through 30G-cannulas at a force of less than 10 Newton. April 4, 2025 7 / 43 Methods The disclosure relates also to a method for producing a product for use as an artificial vitreous body comprising the steps of: a. Provision of a first component comprising tetra-PEG-cyclooctyne and tetra-PEG- azide, wherein tetra-PEG- cyclooctyne is in molar excess as compared to tetra- PEG-azide, wherein in one embodiment the first component comprises tetra-PEG-cy- clooctyne to tetra-PEG-azide in a molar ratio r of at least 0.51, of at least 0.55, of at least 0.60, of at least 0.65, of at least 0.70, or of at least 0.75. In another em- bodiment the first component comprises tetra-PEG-cyclooctyne to tetra-PEG-az- ide in a molar ratio r of up to 0.95, of up to 0.90, of up to 0.85, or of up to 0.80. In yet another embodiment the first component comprises tetra-PEG-cyclooctyne to tetra-PEG-azide in a molar ratio r of from 0.51 to 0.95, of from 0.55 to 0.90, of from 0.60 to 0.85, or of from 0.65 to 0.80, wherein r = [tetra-PEG-cyclooctyne] in mol / ([tetra-PEG-cyclooctyne] in mol + [tetra-PEG-azide] in mol). and b. Provision of a second component comprising tetra-PEG-azide and tetra-PEG- cyclooctyne wherein tetra-PEG-azide is present in molar excess as compared to tetra-PEG-cyclooctyne, wherein in one embodiment the second component comprises tetra-PEG-azide to tetra-PEG-cyclooctyne in a molar ratio s of at least 0.51, of at least 0.55, of at least 0.60, of at least 0.65, of at least 0.70, or of at least 0.75. In another embodi- ment the second component comprises tetra-PEG-azide to tetra-PEG- cy- clooctyne in a molar ratio s of up to 0.95, of up to 0.90, of up to 0.85, or of up to 0.80. In yet another embodiment the second component comprises tetra-PEG-azide to tetra-PEG-cyclooctyne in a molar ratio s of from 0.51 to 0.95, of from 0.55 to 0.90, of from 0.60 to 0.85, or of from 0.65 to 0.80, wherein s = [tetra-PEG-azide] in mol / ([tetra-PEG-azide] in mol + [tetra-PEG- cyclooctyne] in mol), c. Storing the first and the second component separately for at least 1 hour at 20°C; April 4, 2025 8 / 43 d. Optionally, lyophilizing the first and / or the second component. In one embodiment the present disclosure relates also to a product comprising the two compo- nents produced by the before-mentioned method. The first and second component may for a hydrogel when mixed with each other. The hydrogel starts to polymerize after mixing of the two components but remains in soluble form (as a sol) within the first 15 minutes after mixing of the two components. When polymerized it forms the artificial vitreous body. Thus, the present disclosure also pertains to a method for obtaining a hydrogel for use as an ar- tificial vitreous body comprising the steps of the before-mentioned method and, further the steps of a. Optionally, reconstituting the first and / or the second lyophilized component in a sterile buffered saline, b. Mixing the first and the second component in a ratio (vol.-%) from 3:1 to 1:3, from 2:1 to 1:2, from 1.5:1 to 1:1.5, from 1.25:1 to 1:1.25, or of about 1:1, c. thereby obtaining the hydrogel in sol-state. Upon mixing of the two components a hydrogel-formation by “strain-promoted azide-alkyne cy- cloaddition” (SPAAC)-reaction is initiated, which leads to the formation of a hydrogel within 10- 20 minutes at 20°C. Thus, the obtained formulation is injectable at least within the first 5 minutes after mixing, in one embodiment within the first 10 minutes after mixing, within the first 15 minutes after mixing, or within the first 20 minutes after mixing. In one embodiment the pre- sent disclosure relates to a hydrogel comprising the two components produced by the before- mentioned method. Hydrogel properties The hydrogel as disclosed hereinunder may have at least one of the features selected from ^ after polymerization having a polymer content of less than 12.0 g / l, in one embodiment about 10.0 g / l, in another embodiment less than 8.0 g / l, in another embodiment less than 6.0 g / l, in another embodiment less than 4.0 g / l, or in another embodiment less than 3.5 g / l; ^ a pH from 6.5 to 8.5; in one embodiment from 7.0 to 7.4; in one embodiment a pH of 7.4 April 4, 2025 9 / 43 (at a Temperature of 36-37.4°C), and an osmolarity of 300 mOsm / L ^ a gelation time of 20 minutes or less, in one embodiment of 15 minutes or less, in an- other embodiment of 10 minutes or less; ^ being free of reactive groups such as thiols, maleimide, aldehydes, peroxides, thiol-ene (Michael addition), NHS-ester, amines, carboxylates, and / or diamines. ^ having after polymerization a swelling pressure Πsw = Πos – Πel of less than 1 kPa, wherein Πsw is the swelling pressure, Πos is the osmotic pressure, and Πel is the elastic pressure within the hydrogel; ^ being injectable with 27G and / or 30G needles in the sol-state, ^ having at 25°C and more than 20 minutes after mixing, a storage modulus G` of at least 5 Pa, and a loss modulus G`` of less than 0.5 Pa. It is one advantage of the disclosed hydrogels, that they polymerize at physiological condi- tions (i.e. physiological pH, osmolarity, etc.). Thus, they can be administered to the eye with- out any side-effects which may arise from the injection of non-physiological compositions, such as pain or irritations. Ophthalmologic properties of the hydrogel The hydrogel according to the present disclosure has excellent optical properties, which allow ophthalmological examinations, especially fundoscopy. For example, the hydrogel according to the present disclosure has a refractive index of about 1.33 to 1.34, which is similar to that of the natural vitreous body. The hydrogel is optical clear and free of silicone-oil-reflexes, in fact, it is even better with respect to forward light scattering and turbidity as compared to natural vitreous bodies (cf. examples). The hydrogels of the disclosure allow ultrasonic measurements as well as with natural vitreous bodies. Uses In one embodiment the product disclosed herein and / or the hydrogel disclosed herein, may comprise further an active agent for use as a drug-release system in the eye. In one embodiment the active agent may be selected from the group consisting of anti-VEGF drugs such as bevacizumab (Avastin), ranibizumab (Lucentis), aflibercept (Eylea); steroids such April 4, 2025 10 / 43 as triamcinolone acetonide, dexamethasone implant (Ozurdex), antibiotics such as vancomycin, ceftazidime, amikacin, gentamycin, amphotericin b, ciclosporin; other medications such as methotrexate, 5-fluor uracil, and foscarnet; antiviral agents such as acyclovir (Zovirax), valacy- clovir (Valtrex), ganciclovir (Zirgan), voriconazole, trifluridine (Viroptic), cidofovir (Vistide), and famciclovir; nonsteroidal anti-inflammatory drugs (NSAIDs), such as ketorolac (Acular, Acuvail), bromfenac (Prolensa, Bromday), nepafenac (Nevanac), diclofenac (Voltaren, Diclofex), and flurbiprofen (Ocufen); as well as combinations thereof. In one embodiment the product disclosed herein and / or the hydrogel disclosed herein, may be used in the treatment of a condition or disease selected from retinal detachment, diabetic reti- nopathy, macular degeneration, traumatic eye injuries, vitreous hemorrhage, and infections in the eye interior (vitreous). Device In one embodiment the disclosure provides a device for intravitreal injection into the eye, which may comprise a first chamber with the first component as disclosed hereinunder and a second chamber with a second component as disclosed hereinunder. The device of the disclosure may be configured so that the first and the second chamber are fluidly connected or connectable, so that the first and the second component are mixable within the device before and / or during the injection. In one embodiment the device is a cartridge and / or a syringe. Cytotoxicity The starting-monomers, the first and second component, as well as the hydrogel of the present disclosure are non-toxic. That means they are not toxic in an ARPE-19 cell toxicity screen (ARPE-19 cells are a spontaneously arising human RPE cell line with normal karyology which forms polarized epithelial monolayers on porous filter supports). The measurements were performed according to ISO 10993-5 :2009-10 as described in the ex- amples. In short: In order to perform an ARPE-19 cell toxicity screen, ARPE-19 cells are seeded in 96-well plates (105 cells / well) and incubated at 37 °C for 24 h to allow them to reach confluence. Culture me- dia is replaced with serum-free media containing hydrogel at increasing concentrations and in- cubated at 37 °C for 24 h. After 2 hours of PrestoBlue®incubation, cell viability is assessed by measuring the fluorescent signal using a plate reader with a wavelength of 590 nm and an excitation wavelength of 560 April 4, 2025 11 / 43 nm. In accordance with ISO 10993-5, a reduction of cell viability by 30%, a reduction of cell via- bility by 25%, a reduction of cell viability by 20%, a reduction of cell viability by 15%, a reduction of cell viability by 10%, or a reduction of cell viability by 5% was considered a cytotoxic effect. In one embodiment a polymer was considered “non-toxic” when 10.0 g / l resulted in a reduction of cell viability by 30%, a reduction of cell viability by 25%, a reduction of cell viability by 20%, a reduction of cell viability by 15%, a reduction of cell viability by 10%, or a reduction of cell viabil- ity by 5%. Thus, a polymer showing at 10.0 g / l a reduction of cell viability by more than 50%, by more than 45%, by more than 40%, or by more than 30% was considered “toxic”. As such starting-monomers, the first and second component, as well as the hydrogel of the pre- sent disclosure with the content of less than 12.0 g / l, of less than 10.0 g / l, of less than 8.0 g / l, of less than 6.0 g / l, of less than 4.0 g / l, or in another embodiment of less than 3.5 g / l are ”non- toxic”. As such the hydrogels of the present disclosure are superior to existing hydrogels, especially such comprising reactive groups such as thiols, maleimide, aldehydes, peroxides, thiol-ene (Mi- chael addition), NHS-ester, amines, carboxylates, and / or diamines, which turned out to show toxicity in the ARPE-19 cell tests. Hydrogel-formation by SPAAC-reaction The term "hydrogel-formation by SPAAC reaction" refers to the creation of a hydrogel through a specific chemical reaction known as the strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. Hydrogels are three-dimensional networks of hydrophilic polymers that can absorb and retain a significant amount of water. They have applications in various fields, including biomedi- cine, drug delivery, and tissue engineering. The SPAAC reaction is a type of click chemistry, a term used for reactions that are highly selec- tive, efficient, and create minimal byproducts. In the SPAAC reaction, azide and alkyne func- tional groups react with each other to form a triazole linkage. The key feature of SPAAC is that it can occur under mild physiological conditions without the need for copper catalysts, which are often used in other azide-alkyne reactions. The hydrogel-formation by SPAAC reaction involves incorporating azide and alkyne groups into polymer chains or other molecules that can crosslink to form a hydrogel. The azide and alkyne groups on different polymer chains react with each other through SPAAC, resulting in the for- mation of covalent bonds and the development of a hydrogel network. April 4, 2025 12 / 43 The use of SPAAC for hydrogel formation is particularly advantageous in biological and biomed- ical applications because of its biocompatibility and the ability to perform the reaction under mild conditions that are compatible with living systems. This makes it possible to encapsulate and deliver biological molecules, drugs, or cells within the hydrogel matrix without compromising their functionality. Further SPAAC-reactions according to the present disclosure are depicted in the examples. Definitions A “hydrogel” based on polyethylene glycol (PEG) is a three-dimensional network of hydrophilic polymer chains formed by crosslinking PEG molecules. PEG is inherently hydrophilic, and when organized into a hydrogel, it retains the ability to absorb and retain water. This property makes PEG-based hydrogels suitable for various applications, including drug delivery and tissue engi- neering. The term “tetra-PEG” refers herein to a molecule or compound containing four polyethylene gly- col (PEG) units. Polyethylene glycol is a polymer composed of repeating ethylene glycol units. In the context of tetra-PEG, the "tetra-" prefix indicates that there are four PEG units in the mol- ecule. The "eye interior" refers to the inner part of the eye, and the "vitreous" specifically refers to the vitreous humour, a gel-like substance that occupies the space between the lens and the retina in the eye. The eye is divided into various compartments, and the vitreous is found in the poste- rior segment, which makes up the back part of the eye. An “artificial vitreous body”, also known as an artificial vitreous substitute or vitreous implant, is a medical device designed to replace or augment the vitreous humour in the human eye. The vitreous humour is a gel-like substance that fills the space between the lens and the retina in the eye, providing structural support and helping to maintain the shape of the eye. In some eye surgeries, such as vitrectomy, the natural vitreous humour may be removed or partially re- placed. Artificial vitreous bodies can be used to replace the removed vitreous and restore the eye's structure. Artificial vitreous bodies may also be used in the treatment of retinal detach- ment, a condition where the retina pulls away from the underlying tissues. The presence of an artificial vitreous body can help support the retina and improve surgical outcomes. Furthermore, artificial vitreous bodies are sometimes used in research settings to study the effects of different materials on the eye or to develop new surgical techniques. “Cyclooctyne” is a cycloalkyne, which is a type of unsaturated hydrocarbon containing a cyclic ring of carbon atoms with a triple bond. In the case of cyclooctyne, the carbon ring has eight April 4, 2025 13 / 43 atoms, and there is a triple bond between two of the carbon atoms. The molecular formula for cyclooctyne is C₈H₁₂. Cyclooctyne is used hereinunder as a reactant in copper-free click chem- istry reaction, specifically in strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. The advantages of using cyclooctyne in click chemistry is its stability and lack of reactivity under nor- mal physiological conditions. The following variants are comprised hereinunder, if the term “cyclooctyne” is used: ^ Dibenzocyclooctyne (DBCO or DIBAC): This variant of cyclooctyne is highly reactive with azides and has been widely used for bioconjugation and labelling applications. ^ Cyclooctyn-3-ol (COT or C8-ol): COT is another commonly used variant of cyclooctyne for bioorthogonal chemistry. It can react with azides under mild conditions to form tria- zole linkages. ^ Cyclooctyne with electron-withdrawing groups: Introduction of electron-withdrawing groups on cyclooctyne can enhance its reactivity towards azides, leading to faster click reactions. ^ Cyclooctyne with strained rings: Cyclooctynes with strained rings, such as bicy- clo[6.1.0]nonynes, can exhibit even higher reactivity towards azides, enabling faster click reactions. ^ Cyclooctyne derivatives with fluorophores or affinity tags: These variants of cyclooctyne are functionalized with fluorophores or affinity tags, allowing for simultaneous labelling and detection of biomolecules. The term "azide" refers to a chemical functional group containing three nitrogen atoms (N₃). The azide group is represented as -N₃. The key feature of azides in SPAAC reactions is their ability to react with alkynes (containing a carbon-carbon triple bond, represented as -C≡C-) in a selec- tive and efficient manner and under physiological, copper-free conditions. The following variants are comprised hereinunder if the term “azide” is used: ^ Fluorescent azides: Azides functionalized with fluorescent dyes allow for the visualiza- tion and tracking of biomolecules in biological systems. Common examples include az- ides conjugated with fluorophores such as fluorescein, rhodamine, cyanine, or Alexa Fluor dyes. ^ Clickable azides: Azides with additional functional groups, such as alkyne or alkene April 4, 2025 14 / 43 moieties, enable further chemical modification through subsequent click reactions, ex- panding the versatility of SPAAC chemistry. ^ Hydrophilic azides: Azides modified with hydrophilic groups improve solubility and stabil- ity in aqueous environments, making them suitable for applications in physiological con- ditions. "Molar excess" refers herein to the amount by which the quantity of a particular substance ex- ceeds the stoichiometric amount required for a chemical reaction. In other words, it is the sur- plus amount of one reactant in relation to another reactant, expressed in terms of moles. A “molar ratio” hereinunder is a quantitative expression that describes the ratio of moles of one substance in a chemical reaction to the moles of another substance in the same reaction. In this disclosure the molar ratios “r” and “s” are used, wherein ^ r = [tetra-PEG-cyclooctyne] in mol / ([tetra-PEG-cyclooctyne] in mol + [tetra-PEG-azide] in mol); and ^ s = [tetra-PEG-azide] in mol / ([tetra-PEG-azide] in mol + [tetra-PEG- cyclooctyne] in mol). "Sterile buffered saline" refers to a solution that is free from viable microorganisms (sterile) and contains a buffered saline solution. A buffered solution contains a buffer, which is a substance or a combination of substances that helps maintain the pH of the solution within a specific range. Buffers resist changes in pH and stabilize the acidity or alkalinity of the solution. Saline refers to a solution containing salt (sodium chloride) dissolved in water. In the context of sterile buffered saline, the saline component provides an isotonic environment, meaning it has a simi- lar salt concentration to physiological fluids in the human body. The term comprises specific for- mulations, such as "phosphate-buffered saline" (PBS), which includes phosphate buffers in ad- dition to saline. In the context of hydrogels, the terms "sol" and "gel" refer to two different states that the hydro- gel can exist in based on its structural properties and water content. The “sol state” refers to a more fluid or liquid-like state of a hydrogel. In this state, the polymer chains are dispersed in wa- ter, and the hydrogel exhibits a relatively low viscosity and injectable into the eye. The sol state is characterized by a lack of a well-defined three-dimensional network structure. The polymer chains are not extensively crosslinked, allowing them to move more freely within the aqueous medium. Hydrogels in the sol state are typically pourable or injectable liquids. They can be eas- ily manipulated and may flow or conform to the shape of their container. April 4, 2025 15 / 43 The “gel state” refers to a more solid-like or semi-solid state of a hydrogel. In this state, the poly- mer chains are crosslinked, forming a three-dimensional network that traps water molecules. The gel state is characterized by a higher viscosity and a well-defined structure. The crosslinks between polymer chains give the hydrogel its stability and ability to retain water. Hydrogels in the gel state are solid or semi-solid materials. The term “swelling pressure”, in the context of the hydrogels disclosed hereinunder, refers to the internal pressure exerted by the hydrogel as it absorbs and retains water. Hydrogels are three-dimensional networks of polymer chains that have the ability to absorb and hold a signifi- cant amount of water while maintaining their structural integrity. As water is absorbed, the hy- drogel swells, and this swelling can create an internal pressure within the hydrogel matrix. The “swelling pressure” is a result of the osmotic pressure generated by the movement of water molecules into the hydrogel network. When a hydrogel is placed in a water-containing environ- ment, water molecules move into the hydrogel due to osmosis. As the hydrogel swells and ac- commodates the water molecules, the internal pressure increases. Furthermore, during degra- dation processes the osmotic pressure may increase, resulting in increasing swelling pressure, which is not suitable for the use in the eye, since it may lead to complications including pain and even retinal detachment. The hydrogels of the present disclosure are characterized by a very constant swelling pressure over a long period of time. The swelling pressure may be character- ized by the formula Πsw = Πos – Πel, wherein Πsw is the swelling pressure, Πos is the osmotic pres- sure, and Πel is the elastic pressure within the hydrogel. The storage modulus G` describes the energy that is stored in the sample after a force is ex- erted and can be recovered from the sample after the load is relieved. The loss modulus G``, on the other hand, describes the viscous portion of a material. This viscous portion corresponds to the loss portion of the energy, which is converted into heat through internal friction. April 4, 2025 16 / 43 Examples Example 1. Materials for gel synthesis Balanced Sterile Saline Solution (“BSSS” from BVI Medical, Waltham, USA) was purchased. This balanced salt solution has a pH of approximately 7.0. Tetra-PEG-CO and Tetra-PEG-Azide were purchased from JenKem USA (Plano, Texas, USA). Tetra-PEG-CO (4arm PEG Dibenzocyclooctyne) had the characteristics of having a PEG-mo- lecular weight of 10,000 Dalton, the polydispersity of the PEG raw material measured with GPC was ≤ 1.05; and it can be depicted according to formula I: I Tetra-PEG-Azide (4arm PEG Azide) had the characteristics of having a PEG-molecular weight of 10,000 Dalton, the polydispersity of the PEG raw material measured with GPC was ≤ 1.05; and it can be depicted according to formula II: II April 4, 2025 17 / 43 Example 1a. Components A and B Tetra-PEG-CO and Tetra-PEG-Azide were dissolved in balanced sterile salt solution (“BSSS”) at two different concentrations for both reagents, respectively (7.4 g l-1and 12.6 g l-1), the pH was at about 7.0. Subsequently, component A was created by mixing an equal volume of Tetra-PEG-CO (7.4 g l-1) with Tetra-PEG-Azide (12.6 g l-1), the pH was at about 7.0. Component B was created by mixing an equal volume of Tetra-PEG-CO (12.6 g l-1) and Tetra- PEG-Azide (7.4 g l-1) were mixed. Both components were stored at room temperature (20°C) for at least 12 hours. This concluded the pre-gelation process (cf. figure 1). Example 1b. Hydrogel Upon mixing of components A and B, a hydrogel will form within less than 15 minutes at 37°C, thereby undergoing a sol-gel-transition. This results in a hydrogel with a polymer-concentration of 10 g l-1(cf. figure 1). Example 2. Viscoelastic properties A high-rheometer (MCR 302e, Anton Paar, Austria) was used to track the sol-gel-transition of the pregels. After preparation of the rheometer, an equal volume of the two-pregels were mixed and then injected into the gap of the cone-plate setup of the rheometer. Subsequently, the cone was immediately lowered. The oscillatory shear rheological properties, including the storage modulus (G′) and the loss modulus (G′′), during gelation, were measured at 25°C for thirty minutes to determine the sol-gel-transition. A time-sweep was used to track the sol-gel-transi- tion where a constant strain and frequency (1% and 1.0 Hz) is applied over a long time-period. Triplicate measurements were performed. An exemplary result is presented below. Sol-gel-tran- sition for our hydrogel was reached around an optimal of 10 minutes after injection (cf. figure 2). Example 3. Optical analysis For optical evaluation, the hydrogels underwent turbidity measurements and forward light scat- tering measurements. To measure forward light scattering objectively, a modified C-Quant (Oc- ulus GmbH, Wetzlar, Germany) setup developed by our group that was published previously was used 1–3. For turbidity measurements, a TURB 430 IR (Xylem Analytics Germany GmbH, Germany) was used. Turbidity was measured accordingly to ISO 7027, measuring straylight at April 4, 2025 18 / 43 90° with a light wavelength of 860nm. In both optical assessments, the hydrogel reached lower turbidity and lower straylight than the natural vitreous body indicating even better optical clarity of the gel than natural vitreous bodies (cf. figure 3). Example 4. In-vitro feasibility experiments in porcine eyes Porcine eyes were vitrectomized as currently clinically practiced using a 23G-trocar system. Af- ter the removal of the vitreous body and a fluid-air-exchange, the pre-gels (component A and B) were mixed in a syringe and injected into the vitreous cavity. Gelation occurred and the retina was still visible after gelation indicating good gelation and optical clarity in the surgical setting. Example 5. Simulated ageing and viscoelastic properties of the cyclooctine-azid-hydro- gels To test the optical clarity of our hydrogel after exposure to increased temperature, an in vitro model of accelerated aging was applied. This model was previously used for biomaterials in the field of ophthalmology, specifically, intraocular lens research. First, the gels are placed in a fal- con tube, sealed and then moved into a 45°C water bath for 24 hours. After cooling down to room temperature, viscoelastic properties and forward light scattering were measured again. No significant changes were seen, indicating a good long-term stability after gelation (figure 4). Example 6. Cytotoxic test and comparison with prior art hydrogels To assess cell viability, monomer solutions, oligomers, and gels derived from the materials were tested in a PrestoBlue® assay. For the assay, ARPE-19 cells not older than passage 30 were used. “ARPE-19 cells” refers to a specific cell line derived from the human retinal pigment epithelium (RPE). The RPE is a layer of cells located at the back of the eye, between the neural retina and the choroid. The RPE plays a crucial role in supporting and maintaining the health of the photo- receptor cells in the retina. The ARPE-19 cell line is widely used in scientific research, particu- larly in studies related to ocular biology, retinal diseases, and drug development for eye disor- ders. ARPE-19 cells were established from the spontaneously arising retinal pigment epithelial cell line (RPE) obtained from a 19-year-old male donor. ARPE-19 cells are typically cultured in a specific growth medium that supports their viability and growth. These cells are often used in experiments to mimic the behavior of RPE cells in vivo. ARPE-19 cells exhibit characteristics similar to native human RPE cells. They are epithelial in nature and form a monolayer of cells when cultured. These cells are often used as an in vitro April 4, 2025 19 / 43 model to study the physiology and pathology of the human retina. ARPE-19 are very prone to cell death due to toxic substances in the eye. ARPE-19 cells were incubated with the monomer solutions, the oligomers and an extract of the gel in accordance with ISO 10993-5 standards. The incubation was carried out under cell cul- ture conditions (37 °C, 95% ambient air, 5% CO2, 90% humidity). ARPE-19 cells were cultivated in 200 ml Flasks in 10 ml of ARPE-19 complete medium, consisting of 88,7% Dulbecco’s modi- fied eagle‘s medium, 8,9% fetal bovine serum, 1,5% HEPES-Buffer and 0,9% Penicillin / Strepto- mycin. When cell confluency passed 80%, the cells were detached with Trypsin-EDTA. Then, cell count was determined using a Neubauer counting chamber. Subsequently, the Trypsin- EDTA containing medium was removed after centrifugation. The remaining cells were then di- luted with complete medium to a concentration of 106 cells / ml. In a 96-well plate, 105 cells (100 µl) were seeded in each well and left to incubate for 24 h. On the second day, the medium was aspirated, and the cells were washed with 200 μL of phosphate-buffered saline (PBS). Subse- quently, the cells were exposed to 100 μL of the respective treatment for 24 hours. The monomers were dissolved in a solution of 0.5% Tween in BSS at a concentration of 20 g / L. To achieve a final concentration of 10 g / L (equivalent to the maximum concentration that is in- jected), an equal volume of complete medium was added. Concentrations of 5 g / L, 2.5 g / L, 1.25 g / L, 0.63 g / L, 0.31 g / L, 0.16 g / L, 0.08 g / L, 0.04 g / L and 0.02 g / L were prepared by dilution with corresponding amounts of complete medium. The assessment of the gels was performed in ac- cordance with ISO 10993-5 standards. Therefore, an extract of the gels (10 g / L) was prepared by incubating 4.5 mL of gels with 0.5 mL of ARPE Complete Medium at 37°C for 24 hours. The 0.5 mL were then used as a stock solution, which was diluted with complete medium to achieve concentrations 5 g / L, 2.5 g / L, 1.25 g / L, 0.63 g / L, 0.31 g / L, 0.16 g / L, 0.08 g / L, 0.04 g / L and 0.02 g / L.100 µl of each concentration were incubated in ARPE-19-containing wells of a 96-well plate. As a negative control, cells were treated with 1% Triton X-100, while for the positive con- trol, cells were incubated with ARPE complete medium. To simulate long-term conditions, the incubation was carried out for 24 hours. After removing the supernatant and subsequent wash- ing of the wells with 200 µl of PBS on the third day, the cells were incubated for 2 hours with 100 μL of PrestoBlue® solution (diluted 1:10 in medium). To reduce the background fluores- cence signal, blank samples (wells without cells but with 100 μL of PrestoBlue® solution) were carried out. After 2 hours of PrestoBlue® incubation, cell viability was assessed by measuring the fluorescent signal using a plate reader with a wavelength of 590 nm and an excitation wave- length of 560 nm. In accordance with ISO 10993-5, a reduction of cell viability by 30% was con- sidered a cytotoxic effect. Each concentration was incubated in 3 wells of a 96-well plate (n=3). The results of 3 replicate 96-well plates (N=3) were averaged to ensure reproducibility and April 4, 2025 20 / 43 statistical significance. All steps except gel preparation and presto-blue incubation were carried out under aseptic conditions. The monomer solutions showed no toxicity at the highest concentration of 10 g / L with a cell via- bility of 100.07 (± 2.111) % for the tetra-PEG-azide monomers and a cell viability of 102.18 (±1.299) % for the tetra-PEG-DBCO monomers. In comparison the hydrogel itself showed no toxicity at the highest concentration of 10 g / L with a cell viability of 86.85 (±0.631) %, cf. figures 5 - 7. Hydrogels according to Hayashi et al., 2017 (nature biomedical engineering, March 9, 2017, Vol.1) turned out to show a reduction of cell viability of more than 30% at the highest concen- tration of 10 g / L, and therefore are to be considered toxic in the ARPR-19-assay as described. Example 7. Formation of the oligomeric pregels as determined by Nanodrop absorption Formation of the oligomeric pregels as determined by Nanodrop absorption measurements is completed after roughly 3 hours compared to previous literature by Hayashi et al suggesting up to 12 hours. Free DBCO groups are responsible for absorption. No chance in absorption is visi- ble after 120-240 minutes (cf. also Figure 8). Viscoelastic and optical properties of the hydrogel are stable after 3 months incubation in bal- anced salt solution Viscoelastic and optical properties were stable in balanced salt solution for a 12-week incuba- tion time at 8°C. Straylight remained between 0.5-1 deg2 / sr and thus within the limit of the hy- drogel directly after formation. The previously measured viscoelastic were comparable to visco- elastic properties measured directly after gelation. Example 8. Frequency Sweep after 3 months of incubation in balanced salt solution Liposomes and PLGA-nanoparticles can be incorporated into the hydrogel without lowering opti- cal quality and non-clinically relevant increase in straylight (cf. Figure 9). Optical quality was measured using a TriOptics optical bench setup as well as a modified C-Quant setup to measure forward light scattering. Up to concentrations of 0.5 mg / ml were tested for both, liposomal and PLGA-nanoparticle formulations. The impact on the optical properties of the hydro- gels are within the tolerable range. These concentrations of nanoparticles and liposomes can carry a relevant amount of drug. Example 9. Optical impact of liposomes and PLGA-nanoparticles incorporated into the April 4, 2025 21 / 43 hydrogel platform Optical impact of liposomes and PLGA-nanoparticles incorporated into the hydrogel platform was measured in different experiments (cf. figure 10): A) Straylight increases with increasing concentration of nanoparticles but stays within a tolera- ble area B) Liposomal and C) PLGA-nanoparticles from left to right with 0.1, 0.2, 0.3, 0.4 and 0.5 mg / ml concentration do not lead to significant changes of the USAF-target D) Modulation transfer function shows no change from baseline optical performance. The corresponding characteristics of PLGA-nanoparticles und liposomes are depicted in figure 11. For the above-mentioned concentration, storage and loss modulus remained within target range for all tested frequencies (cf. figure 12). Example 10. Cryo-SEM Cryo-SEM imaging showed no chance in the pore size and network pattern when nanoparticles or liposomes are added (exemplary images of unloaded gels and a gel loaded with 1 mg / ml lipo- somes) as showcased in figure 13. Drugs can be incorporated in the hydrophilic media and allow the sustained release from the hydrogel >7 days in vitro, Antibody drugs are still functional after contact with the hydrogel in contrast to currently used clinical tamponades. The measured amounts of released API over 120 h are summarized in Table 1. All concentrations were analyzed, however the total amount of released voriconazole (0.1 mg per eye) was below the lower limit of quantification and is therefore not further discussed. Over a period of 120 h and regarding the median value, the hydrogel released about 35 % of the incorporated drugs independently of their chemical nature. However, voriconazole as a very lipo- philic drug was released noticeably slower with a percentage of around 22 % after 120 h com- pared to the other hydrophilic APIs. For the studied small molecules, the release increased with a higher loading dose, which was rather the opposite for bevacizumab. The exchange of the supernatant every 24 h induced a faster API release that is thought to be caused by an induced higher concentration gradient. April 4, 2025 22 / 43Table 1 - Amounts of API over 120hDose per Bevacizumab Ceftazidime Vancomycin Voriconazole eye [mg] 2 10 2 20 1 10 0.1 1 Timepoint Release [%] [h] 6 6.58 ± 3.92 ± 6.98 ± 7.25 ± 5.65 ± 5.35 ± 4.18 ± 0.00 0.01 0.00 0.00 0.00 0.01 0.01 24 9.79 ± 6.80 ± 8.92 ± 9.78 ± 7.33 ± 7.78 ± 5.64 ± 0.00 0.01 0.00 0.00 0.00 0.00 0.01 30 13.51 ± 8.71 ± 14.25 ± 16.11 ± 12.27 ± 12.69 ± 9.04 ± 0.00 0.02 0.00 0.01 0.01 0.00 0.01 48 16.00 ± 11.94 ± 16.12 ± 17.53 ± 13.76 ± 14.37 ± 10.40 ± 0.01 0.02 0.00 0.00 0.01 0.00 0.00 54 18.73 ± 16.14 ± 21.20 ± 22.52 ± 18.52 ± 19.74 ± 13.24 ± 0.01 0.04 0.00 0.00 0.02 0.01 0.00 <LLOD* 72 20.89 ± 18.17 ± 22.63 ± 24.72 ± 19.74 ± 21.12 ± 14.45 ± 0.02 0.05 0.00 0.01 0.02 0.01 0.00 78 24.58 ± 22.82 ± 27.96 ± 30.05 ± 25.28 ± 26.94 ± 17.63 ± 0.03 0.08 0.01 0.02 0.03 0.02 0.01 96 27.71 ± 24.57 ± 28.94 ± 31.17 ± 26.31 ± 28.35 ± 18.09 ± 0.04 0.09 0.01 0.01 0.03 0.03 0.01 102 31.68 ± 28.81 ± 34.71 ± 36.36 ± 32.26 ± 35.13 ± 21.95 ± 0.06 0.12 0.02 0.02 0.04 0.03 0.01 120 35.06 ± 30.61 ± 35.01 ± 37.45 ± 32.71 ± 35.59 ± 22.36 ± 0.07 0.12 0.02 0.02 0.03 0.03 0.01 April 4, 2025 23 / 43 Example 11. PEG2000 – in-vivo biocompatibility in landrace pigs 3 landrace pigs were injected with 0.1 ml at 400mg / ml of PEG 2000 to establish the baseline biocompatibility of PEG. No signs of cytotoxicity, including electroretinography and fundus pho- tography, were seen. Results are presented in figures 14 and 15. April 4, 2025 24 / 43

Claims

Claims 1. Product comprising a first and a second component as a combined preparation for simul- taneous or sequential use as an artificial vitreous body, a. wherein the first component comprises tetra-PEG-cyclooctyne and tetra-PEG- azide, wherein tetra-PEG-cyclooctyne is present in molar excess as compared to tetra-PEG-azide, and b. wherein the second component comprises tetra-PEG-azide and tetra-PEG-cy- clooctyne wherein tetra-PEG-azide is present in molar excess as compared to tetra-PEG-cyclooctyne.

2. The product according to claim 1, a. wherein in the first component comprises tetra-PEG-cyclooctyne in a molar ratio r of at least 0.51 and tetra-PEG-azide in a molar ratio r of 1-r, wherein r = [tetra-PEG-cyclooctyne] in mol / ([tetra-PEG-cyclooctyne] in mol + [tetra-PEG-azide] in mol), and b. wherein in the second component comprises tetra-PEG-azide in a molar ratio s of at least 0.51 and tetra-PEG-cyclooctyne in a molar ratio s of 1-s, wherein s = [tetra-PEG-azide] in mol / ([tetra-PEG-azide] in mol + [tetra-PEG- cy- clooctyne] in mol).

3. The product according to claim 1 or 2, wherein the first and / or second component is ly- ophilized.

4. A method for producing a product for use as an artificial vitreous body comprising the steps of: a. Provision of a first component comprising tetra-PEG-cyclooctyne and tetra-PEG- azide, wherein tetra-PEG-cyclooctyne is in molar excess as compared to tetra- PEG-azide, and b. Provision of a second component comprising tetra-PEG-azide and tetra-PEG- cyclooctyne wherein tetra-PEG-azide is present in molar excess as compared to April 4, 2025 25 / 43tetra-PEG-cyclooctyne, c. Storing the first and the second component separately for at least 1 hour at 20°C; d. Optionally, lyophilizing the first and / or the second component.

5. A product produced by the method according to claim 4.

6. A method for obtaining a hydrogel for use as an artificial vitreous body comprising the steps of claim 4 and, further the steps of e. Optionally, reconstituting the first and / or the second lyophilized component in a sterile buffered saline, f. Mixing the first and the second component in a ratio (vol.-%) from 3:1 to 1:3, pref- erably in a ratio (vol.-%) of about 1:1, g. thereby obtaining the hydrogel in sol-state.

7. A hydrogel produced by the method according to claim 6.

8. The hydrogel according to claim 7, having at least one of the features selected from ^ a polymer content of less than 12.0 g / l, preferably about 10.0 g / l; ^ a pH from 6.5 to 8.5; ^ a gelation time of 15 minutes or less; ^ being free of reactive groups such as thiols, maleimides, aldehydes, peroxides, thiol- enes, NHS-ester, amines, carboxylates, and / or diamines; ^ having a swelling pressure Πsw = Πos – Πel of less than 1.0 kPa, wherein Πsw is the swelling pressure, Πos is the osmotic pressure, and Πel is the elastic pressure within the hydrogel.

9. An artificial vitreous body comprising the hydrogel of claim 7 or 8.

10. The product according to claim 1, the product according to claim 5, or the hydrogel ac- cording to claim 7 comprising further an active agent for use as a drug-release system in the eye.

11. The product or the hydrogel according to claim 10 for use in the treatment of a condition April 4, 2025 26 / 43or disease selected from retinal detachment, diabetic retinopathy, macular degeneration, traumatic eye injuries, vitreous haemorrhage, and infections in the eye interior (vitreous).

12. The product or the hydrogel according to claims 10 or 11, wherein the active agent is se- lected from anti-VEGF drug, steroids, antibiotics, antiviral agents, nonsteroidal anti-in- flammatory drugs (NSAIDs), other medication such as methotrexate, 5-fluor uracil, and / or foscarnet, as well as combinations thereof.

13. The product or the hydrogel according to claim 12 , wherein the anti-VEGF drug is se- lected from the group consisting of bevacizumab, ranibizumab, and aflibercept; the ster- oid is selected from the group consisting of triamcinolone acetonide, and dexame- thasone implant, the antibiotic is selected from the group consisting of vancomycin, ceftazidime, amikacin, gentamycin, amphotericin b, and ciclosporin, the antiviral agent is selected from the group consisting of acyclovir, valacyclovir, ganciclovir, trifluridine, cidofovir, and famciclovir; the nonsteroidal anti-inflammatory drug (NSAID) is selected from the group consisting of ketorolac, bromfenac, nepafenac, diclofenac, and flurbi- profen, as well as combinations thereof.

14. A device for intravitreal injection into the eye, comprising a first chamber with a first com- ponent and a second chamber with a second component, a. wherein the first component in the first chamber comprises tetra-PEG-cy- clooctyne and tetra-PEG-azide, wherein tetra-PEG-cyclooctyne is present in mo- lar excess as compared to tetra-PEG-azide, and b. wherein the second component in the second chamber comprises tetra-PEG-az- ide and tetra-PEG-cyclooctyne wherein tetra-PEG-azide is present in molar ex- cess as compared to tetra-PEG-cyclooctyne.

15. The device according to claim 14, wherein the first and the second chamber are fluidly con- nected or connectable, so that the first and the second component are mixable within the device before and / or during the injection. April 4, 2025 27 / 43

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