Method for preparing crosslinked microparticles, microparticles obtained thereby and their use

Abstract

The present invention generally relates to the field of dermal fillers, and more specifically to a method for preparing a microparticles comprising crosslinked sulfated glycosaminoglycan (GAG) polysaccharide moieties. The present invention further relates to microparticles obtainable by said method and their use, as well as compositions comprising said microparticles and uses thereof.

Description

Merz Aesthetics GmbH 120725P502PCNovember 7, 2025METHOD FOR PREPARING CROSSLINKED MICROPARTICLES, MICROPARTICLES OBTAINED THEREBY AND THEIR USEFIELD OF THE INVENTION

[0001] The present invention generally relates to the field of dermal fillers, and more specifically to a method for preparing microparticles comprising crosslinked sulfated glycosaminoglycan (GAG) polysaccharide moieties. The present invention further relates to microparticles obtainable by said method and their use, as well as compositions comprising said microparticles and uses thereof.BACKGROUND OF THE INVENTION

[0002] Nonsurgical rejuvenation procedures using injectable dermal fillers are currently considered to be the second most common non-surgical aesthetic treatments performed worldwide, behind botulinum toxin injections. The practitioners using such dermal fillers typically desire that such fillers do not provoke toxic or immunologic adverse effects when administered under the conditions of interest, show good biocompatibility, are injectable without burden and based on natural materials. Furthermore, such dermal fillers should remain in a spatially defined area and have a sufficient stability in biological systems such as when being injected. Of the numerous filler types available to the clinician, hyaluronic acid is by far the most commonly utilized filler material today.

[0003] Hyaluronic acid (HA) has a relatively good biological acceptability. However, HA is rapidly degraded in vivo by enzymatic and free radical degradation, and it has an in vivo halflife of only about 24-48 hours, which results in the filler material not being suitable for longterm solutions. Furthermore, storability and shelf life of unmodified polysaccharides, such as HA, is often limited, due to partial degradation and a resulting decrease in the products viscosity. Therefore, in most commercial products, hyaluronic acid is crosslinked to increase its longevity. While many different crosslinking agents have been investigated and used inMerz Aesthetics GmbH 120725P502PCNovember 7, 2025 crosslinked hyaluronic acid products, 1 ,4-butanediol diglycidyl ether (BDDE) is today the most commonly used crosslinking agent.

[0004] Under alkaline reaction conditions, the epoxide groups of BDDE preferentially react with the primary hydroxyl group of HA to form stable ether bonds. As a result, the HA chains are linked to form a stable three-dimensional network structure. However, BDDE has some disadvantages. For example, any BDDE-crosslinked HA product will always contain unreacted (i.e. "free") BDDE as well as partially hydrolyzed BDDE (i.e. epoxydioles; EPD) and BDDE degradation products. BDDE and EPD contain reactive epoxide groups and are therefore generally considered toxic, and BDDE is even suspected of being carcinogenic. Therefore, a laborious purification procedure needs to be set up to remove these impurities to the greatest possible extent. Furthermore, BDDE is a small diepoxide molecule with no variability in its structure. Consequently, it is only possible to influence the properties of the final gel product (e.g., strength and cohesivity) via the amount of BDDE and the degree of crosslinking.

[0005] WO 2011 / 1194568 A1 discloses cohesive soft tissue fillers, for example, dermal and subdermal fillers, based on hyaluronic acids and optionally including proteins. The hydrogel is suitable for soft-tissue augmentation and comprises a crosslinked biocompatible polymer having zero-length crosslinked moieties and optionally at least one other active ingredient incorporated into said crosslinked biocompatible polymer. The hydrogel is made by providing at least one crosslinkable biocompatible polymer, dissolving said at least one crosslinkable biocompatible polymer in a buffered solution wherein said buffered solution maintains a pH from approximately 6.0 to 8.0, adding at least one zero-length crosslinking agent to said buffered solution to form a reaction mixture, and allowing said reaction mixture to stand for a time appropriate to form the hydrogel.

[0006] WO 2016 / 096594 A1 discloses a cosmetic process for caring for the skin comprising the topical application to the skin of a cosmetic composition comprising a grafted polysaccharide polymer (I) and exposure of the treated skin to light radiation, polymer (I) being of formula: PS-(CO-NH-L-X)a(COOH)b in which PS denotes the basic backbone of theMerz Aesthetics GmbH 120725P502PCNovember 7, 2025 polysaccharide bearing the carboxylic acid groups; L is a divalent hydrocarbon-based group containing from 1 to 20 carbon atoms; X denotes a photoactive group of azide or diazirine type; a denotes the content of COOH groups substituted with the group -NH-L-X; b denotes the content of unsubstituted free COOH groups; a being between 0.01 and 0.8; b being between 0.2 and 0.99; a + b = 1 .

[0007] WO 2016 / 096594 A1 also relates to the polymers (I) bearing a photoactive group X of diazirine type and to a composition comprising such a polymer in a physiologically acceptable medium. The polysaccharide bearing a carboxylic acid group may be chosen from hyaluronic acid, chondroitin, chondroitin sulfate, alginic acid, heparin, heparin sulfate and xanthan gum. The grafted polysaccharide used my be prepared especially by activation of the carboxylic acid groups by reaction with a carbodiimide such as 1 -ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride or a dialkoxytriazine such as 4-(4,6- dimethoxy-1 ,3,5-triazin-2-yl)-4-methylmorpholinium chloride to form an intermediate compound, followed by reaction with an amine of formula X-L-NH2 to form compound (I).

[0008] WO 2017 / 114861 A1 discloses a method for at least partial deacetylation of a biopolymer comprising acetyl groups, comprising: a1) providing a biopolymer comprising acetyl groups; a2) reacting the biopolymer comprising acetyl groups with hydroxylamine (NH2OH) or a salt thereof at a temperature of 100 °C or less for 2-200 hours to form an at least partially deacetylated biopolymer; and a3) recovering the at least partially deacetylated biopolymer. The hydrogel may be made of crosslinked glycosaminoglycans, particularly crosslinked hyaluronic acid, chondroitin or chondroitin sulfate. An activation step b) and a crosslinking step c) may occur simultaneously or the activation step b) may occur prior to and separately from the crosslinking step. The coupling agent of step b) may be a peptide coupling agent selected from the group consisting of triazine-based coupling reagents, preferably 4- (4,6-dimethoxy-1 ,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) and 2-chloro- 4,6-dimethoxy-1 ,3,5-triazine (CDMT), carbodiimide coupling reagents, preferably N-(3- dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) combined with N-hydroxysuccinimide (NHS), imidazolium-derived coupling reagents, Oxyma and COMU.Merz Aesthetics GmbH 120725P502PCNovember 7, 2025

[0009] WO 2022 / 237901 A1 discloses hyaluronic acid compositions comprising hyaluronic acid or modified hyaluronic acid or crosslinked hyaluronic acid and slowly resorbable particles, spheres and granules. The composition may comprise a crosslinked hyaluronic acid, or a cohesive crosslinked HA carrier produced by crosslinking hyaluronic acid with by divinylsulfone in alkaline environment, and then precipitated the crosslinked hyaluronic acid with ethanol; and PLA microspheres, PEG-PLA microspheres, PLA-PCL microspheres, or crosslinked HA microspheres produced by emulsified crosslinking reaction using £-polylysine and 4-methylmorpholine hydrochloride (DMTMM) as the crosslinker in the presence of organic oil (such as olive oil or silicone oil) under stirring. The composition may further comprise additives, for example those selected from the group consisting of: local anesthesia drugs such as lidocaine, procaine, and / or polyols stabilizers, such as glycerin, mannitol, butanediol, sorbitol, and / or a stabilizer with chelating ability, such as EDTA, EGTA, citric acid, sodium citrate, and / or a sulfur stabilizer or dissolution promotor, such as Chondroitin Sulfate Sodium (CS), Glucosamine Sulphate (GS) or Methyl sulfonyl methane (MSM) , and / or wherein soluble small molecules are added through dialysis process.

[0010] WO 2022 / 268871 A1 discloses a further method for preparing a cross-linked material comprising forming amide bonds that conjugate one or more fibroin moieties with one or more polysaccharide moieties. In addition, US 2023 / 0172970 A1 discloses crosslinked chondroitin sulfate particles. The crosslinking may be performed with divinyl sulfone (DVS), a bis-epoxide such as butanediol diglycidyl ether ("BDDE"), 1 ,2,3,4-diepoxybutane, 1 , 2,7,8- diepoxyoctane, polyethylene glycol)diglycidyl ether, or a bis-carbodiimide like p-phenylene BDCI.

[0011] Notwithstanding recent advances, there is an ongoing interest in filler designs to meet specific cosmetic needs. In other words, there is a continuing need for improvements to and / or alternative formulations for crosslinked dermal fillers and / or novel delivery systems that address the specific requirements necessary for a particular cosmetic use. Furthermore, there is an unmet need for an efficient method for preparing said crosslinked materials.Merz Aesthetics GmbH 120725P502PCNovember 7, 2025OBJECTS OF THE INVENTION

[0012] Having regard to the above, it is an object of the present invention to provide a method for the preparation of a crosslinked material for skin regeneration, which not only has a high stability but can also be rapidly degraded in case of complications, and which method allows for the tuning of the rheological properties of the crosslinked material.SUMMARY OF THE INVENTION

[0013] The above object is achieved by the provision of a two-phase crosslinking procedure resulting in the size-controllable preparation of stable microparticles comprising crosslinked sulfated glycosaminoglycan (GAG) polysaccharide moieties.

[0014] In a first aspect, the present invention provides a method for preparing a microparticle comprising crosslinked sulfated GAG polysaccharide moieties, the method comprising the steps of:(a) providing a polar composition comprising a polar solvent and sulfated GAG polysaccharide moieties, wherein the sulfated GAG polysaccharide moieties comprise carboxylic acid groups and nucleophilic groups,(b) providing a non-polar composition comprising a non-polar solvent,(c) mixing the polar composition and the non-polar composition to obtain an emulsion comprising microparticles comprising sulfated GAG polysaccharide moieties, wherein the emulsion is a reverse emulsion,(d) adding a carboxyl-activating agent to the emulsion obtained in step (c) to activate at least some of the carboxylic acid groups,(e) allowing reaction of at least some of the activated carboxylic acid groups of the sulfated GAG polysaccharide moieties with at least some of the nucleophilic groups of the sulfated GAG polysaccharide moieties to obtain microparticles comprising crosslinked sulfated GAG polysaccharide moieties, andMerz Aesthetics GmbH 120725P502PCNovember 7, 2025(f) purifying the microparticles comprising crosslinked sulfated GAG polysaccharide moieties.

[0015] The sulfated GAG polysaccharide moieties have a relatively high solubility in the polar composition and at the same time ensure a sufficiently low viscosity for the formation of microparticles, in particular spherical microparticles, in particular microspheres, since a relatively high viscosity, as is the case with hyaluronic acid polysaccharides having a relatively long polymer chain, would prevent the formation of microparticles, in particular spherical microparticles, in particular microspheres. Furthermore, the viscosity and stability of said microparticles may be widely maintained by using sulfated GAG polysaccharide moieties. The above method can be conducted without undue burden and with relatively low efforts, since it essentially requires one educt, namely the sulfated GAG polysaccharide moieties, in addition to two solvents and a carboxyl-activating agent. Further components, such as linkers are not required.

[0016] Furthermore, by crosslinking of at least some of the carboxylic acid groups of the sulfated GAG polysaccharide moieties with at least some of the nucleophilic groups of the sulfated GAG polysaccharide moieties, the following is enabled: a covalent linkage, in particular a covalent linkage via ester bonds, between at least some of the carboxylic acid groups of the sulfated GAG polysaccharide moieties and at least some of the nucleophilic groups, in particular at least some of the hydroxy groups, of the sulfated GAG polysaccharide moieties without an interconnecting linker structure, in particular without an interconnecting linker structure derived from the carboxyl-activating agent. This distinguishes the method according to the invention from methods in which for example divinyl sulfone or BDDE are used, which results in a xenobiotic linker structure being introduced between the cross-linked moieties. These xenobiotic linker structures are chemical structures that are extrinsic to the normal metabolism of an organism, in particular a human organism, and would reach toxic concentrations without metabolism. Thus, by avoiding xenobiotic linker structures, the method according to the invention allows the preparation of microparticles comprising crosslinked polysaccharide moieties with an improved safety profile due to a reduced quantity of xenobiotic structures.Merz Aesthetics GmbH 120725P502PCNovember 7, 2025

[0017] Moreover, in the method according to the invention, after introduction of a covalent linkage between at least some of the carboxylic acid groups of the sulfated GAG polysaccharide moieties and at least some of the nucleophilic groups of the sulfated GAG polysaccharide moieties in step (e), an improved purification is enabled in step (f) since all sub-products resulting from the activation reaction can be easily removed and do not stay as pending groups in the reaction mixture. This results in very low amounts of unwanted impurities and further improves the safety profile of the purified product.

[0018] In a second aspect, the present invention provides a microparticle obtainable by the method according to the first aspect of the present invention. The thus obtained microparticle contains very low amounts of unwanted impurities like residual non-reacted carboxyl- activating agents or partially reacted carboxyl-activating agents or degradation products thereof. Thus, the microparticle exhibits a very favorable safety profile. The obtained microparticles according to the second aspect of the present invention may have the same properties as the microparticles according to the following third aspect of the invention.

[0019] In a third aspect, the present invention provides a microparticle comprising sulfated glycosaminoglycan (GAG) polysaccharide moieties, wherein the sulfated GAG polysaccharide moieties are covalently crosslinked with each other by ester bonds between at least some of the carboxylic acid groups of the sulfated GAG polysaccharide moieties and at least some of the nucleophilic groups of the sulfated GAG polysaccharide moieties, in particular without an interconnecting linker structure.

[0020] Due to above-indicated cross-linking via the sulfated GAG polysaccharide moieties, the microparticles are characterized by an improved stability and have excellent properties for the skin, which makes the microparticles suitable to promote fibroblast proliferation and improve skin quality. In addition, it has been found that the viscosity of said microparticles may be widely maintained and said microparticles may have a long-lasting stability. Likewise, also enzymatic degradation may be desirably diminished.Merz Aesthetics GmbH 120725P502PCNovember 7, 2025

[0021] In a fourth aspect, the present invention provides the microparticles according to the second and / or third aspect of the present invention for use as a medicament, in particular for the treatment of degenerative joint diseases, in particular osteoarthritis, or connective tissue diseases, in particular rheumatoid arthritis. The microparticles according to the second and / or third aspect of the present invention may be used as a drug delivery system. The drug delivery system comprises a microparticle as described above and at least one polar cargo molecule, in particular a drug, encapsulated in the microparticles. In particular, the drug delivery system is for use as a medicament.

[0022] In a fifth aspect, the present invention provides a hydrogel comprising a microparticle according to the second and / or third aspect of the present invention, an aqueous solution, and optionally at least one polar cargo molecule, wherein the at least one polar cargo molecule is encapsulated in the microparticle. Thus, a novel delivery system for polar compounds can be provided by the hydrogel comprising microparticles with a polar cargo molecule encapsulated in the microparticle. Furthermore, due to the microparticles according to the second and / or third aspect of the present invention comprising crosslinked polysaccharide moieties, a sustained release of the polar cargo molecule encapsulated in the microparticle is enabled. The polar cargo molecule may be a hydrophilic cargo molecule.

[0023] In a sixth aspect, the present invention provides a use of the microparticles according to the second and / or third aspect or the hydrogel according to the fifth aspect of the present invention, for cosmetic applications.

[0024] Non-limiting cosmetic applications include cosmetic treatments of facial lines, facial wrinkles, glabellar lines, nasolabial folds, marionette lines, buccal commissures, peri-lip wrinkles, crow’s feet, subdermal support of the brows, malar and buccal fat pads, tear troughs, nose, lips, cheeks, peroral region, infraorbital region, facial asymmetries, jawlines, and chin.

[0025] Preferred embodiments of the present invention are set forth in the appended claims. Further embodiments and other objects, advantages and features of the presentMerz Aesthetics GmbH 120725P502PCNovember 7, 2025 invention will become apparent from the following detailed description of the invention and the examples.DETAILED DESCRIPTION OF THE INVENTION

[0026] It was surprisingly found that microparticles comprising crosslinked sulfated GAG polysaccharide moieties having beneficial properties can be obtained from a method comprising the reaction to covalent bonds, in particular covalent ester bonds, between at least some of the carboxylic acid groups of the sulfated GAG polysaccharide moieties with at least some of the nucleophilic groups, in particular hydroxy groups, of the sulfated GAG polysaccharide moieties without an interconnecting linker structure between said carboxylic acid groups and said nucleophilic groups due to the use of a carboxyl-activating agent. Viscosity may be widely maintained and enzymatic degradability may be desirably diminished.

[0027] Since the carboxyl-activating agent is not part of the interconnecting linker structure between said carboxylic acid groups and said nucleophilic groups, xenobiotic linker structures as commonly used in the prior art can be avoided. The microparticles comprising crosslinked sulfated GAG polysaccharide moieties may essentially consist of polysaccharide moieties, which can also be found in nature. Since xenobiotic linker structures are avoided in said microparticles, the administration of higher amounts of material to a subject, in particular by injection, may be enabled.

[0028] Advantageously, the microparticles obtainable from the method according to the invention have a long-lasting stability and have longevity in a subject’s body after administration. In addition, the microparticles obtainable from the method according to the invention comprising crosslinked sulfated GAG polysaccharide moieties show a relatively high thermal stability, in particular a thermal stability comparable to microparticles comprising other crosslinked GAG polysaccharide moieties, in particular comparable to microparticles comprising crosslinked hyaluronic acid moieties.Merz Aesthetics GmbH 120725P502PCNovember 7, 2025

[0029] Furthermore, the microparticles comprising crosslinked sulfated GAG polysaccharide moieties have good shear-thinning properties. The microparticles comprising crosslinked sulfated GAG polysaccharide moieties may also have thixotropic properties, and thus may be less viscous when stressed. The microparticles can be easily injected, while still being rather viscous in its target area (when, e.g., administered in a subcutaneous area). With the microparticles comprising crosslinked sulfated GAG polysaccharide moieties, gels, in particular hydrogels, of high viscosity and low extrusion forces can be obtained. In aqueous environment, the microparticles comprising crosslinked sulfated GAG polysaccharide moieties generally form a hydrogel. The microparticles and / or gels, in particular hydrogels, of the present invention can also be used as a super-volumizer.

[0030] In addition, the microparticles comprising crosslinked sulfated GAG polysaccharide moieties can provide excellent properties to the skin. Due to the presence of sulfated GAG polysaccharide moieties crosslinked without an interconnecting linker structure, a mimicking of the extracellular matrix (ECM) is enabled, which may result in the induction of cell proliferation and / or cell migration and / or may serve as a scaffold for cells. In this regard, the microparticles comprising crosslinked sulfated GAG polysaccharide moieties may be used for biostimulation and to improve skin quality (glow, texture, etc.). Thus, the microparticles comprising crosslinked sulfated GAG polysaccharide moieties may be used as a filler, e.g. a dermal filler, which may be populated by cells.

[0031] Within the context of the present invention, chemical constituents of the compositions, such as the microparticles, the hydrogel or the dermal filler, mentioned in singular forms are to be understood as a plurality of molecules of these constituents. For example, the term "allowing reaction of at least some of the activated carboxylic acid groups of the sulfated GAG polysaccharide moieties with at least some of the nucleophilic groups of the sulfated GAG polysaccharide moieties” means that a plurality of molecules of the sulfated GAG polysaccharide moieties are reacted with a plurality of molecules of other sulfated GAG polysaccharide moieties after activation of a plurality of carboxyl groups by a plurality of molecules of the carboxy l-activati ng agent. Furthermore, the term “wherein the polar composition further comprises at least one polar cargo molecule” means that the polarMerz Aesthetics GmbH 120725P502PCNovember 7, 2025 composition further comprises a plurality of molecules of the at least one polar cargo molecule.

[0032] As used herein, the term “microparticle(s)” generally relates to particles that are substantially circular or spherical. In addition, the average diameter of microparticles in general is ranging from 1 micrometers (pm) to 1000 pm, in particular 5 pm to 500 pm, in particular 10 pm to 250 pm, in particular 15 pm to 100 pm, in particular 20 pm to 50 pm. In this context, the term “microsphere”, as used herein, usually refers to spherical particles made up of biodegradable and biocompatible polymers having the size ranging from 1 to 1000 pm, in particular s pm to 500 pm, in particular 10 pm to 250 pm, in particular 15 pm to 100 pm, in particular 20 pm to 50 pm, and optionally incorporating drugs and other bioactives within their core. Since the microparticles as specified according to the invention comprise or consist of biodegradable and biocompatible polymers, namely sulfated glycosaminoglycan (GAG) polysaccharides, and are spherical, and may optionally contain bioactive molecules or drugs, the terms "microparticles" and "microspheres" are used interchangeably in the context of this application.

[0033] The term “ester bond”, as used herein, may be understood in the broadest sense as generally understood in the art. Typically, an ester bond has the structure -O-CO- or - CO-O-, including tautomeric structures thereof.

[0034] The term "hydrogel", as used herein, usually refers to a material having fluidity at room or body temperature between that of a liquid and solid and is capable of absorbing water. The hydrogel is preferably "cohesive", which means that the gel has the capacity not to dissociate because of the affinity of its molecules for each other. Cohesivity is important with regard to gel integrity and, for the purpose of the present invention, can be determined using the Gavard-Sundaram Cohesivity (GSC) scale (Sundaram et al., Plast. Reconstr. Surg. 136:678-686, 2015).

[0035] As used herein, the term "dermal filler" generally refers to a material designed to add volume to, or replace or augment volume of, soft tissue areas of skin. The dermal fillerMerz Aesthetics GmbH 120725P502PCNovember 7, 2025 described herein is normally sterile and generally injectable, i.e. can be dispensed from syringes or similar devices under normal conditions under normal pressure to the desired target site (e.g., in the dermis and hypodermis).

[0036] In the context of the present invention, the term “sulfated glycosaminoglycan (GAG) polysaccharide moieties” is further specified as structures comprising carboxylic acid groups or salts thereof and nucleophilic groups. Thus, the sulfated GAG polysaccharide moieties refer to sulfated GAG polysaccharides comprising carboxylic acid groups or salts thereof and nucleophilic groups. In this regard, the terms “polysaccharide moieties” and “polysaccharides” are used interchangeably in the context of this application. Preferably, the sulfated GAG polysaccharide moieties as specified according to the invention may be selected from the group consisting of chondroitin sulfate and / or derivatives thereof, dermatan sulfate, heparin, heparan sulfate and mixtures thereof. Furthermore, the terms “chondroitin sulfate”, “dermatan sulfate”, “heparin” and “heparan sulfate”, as used herein, respectively include any of their salts, such as sodium salts, potassium salts, lithium salts, magnesium salts, calcium salts, or combinations thereof. For example, the term “chondroitin sulfate” includes chondroitin sulfate and any of its chondroitin sulfate salts, such as salts of chondroitin sulfate and sodium, potassium, lithium, magnesium, calcium, or combinations thereof.

[0037] As exemplarily shown for chondroitin sulfate, it may be a structure comprising the following repeating / monomeric units in an aqueous environment (counterions are not depicted and may be any cations such as, e.g. sodium):

[0038] In the context of the present invention, the sulfated GAG polysaccharide moieties usually refer to polysaccharides composed of repeating disaccharides esterified with sulfuric acid. The individual disaccharide units consist of a uronic acid (usually glucuronic acid, moreMerz Aesthetics GmbH 120725P502PCNovember 7, 2025 rarely iduronic acid, uronic acid of idose), which are linked 1 -3-glycosidically with an amino sugar such as N-acetylglucosamine. The disaccharide units of the chains themselves are linked 1 -4-glycosidically. The sulfated GAG polysaccharide moieties may be one or more types of polysaccharide moieties. These may be different in molecular size and / or may be different in chemical structure.

[0039] The sulfate groups of the sulfated GAG polysaccharide moieties result in the polar composition as specified according to the invention to have a relatively high solubility and a relatively low viscosity. These effects are mainly enabled by the negative charge of the sulfate group, in particular the negative net charge of -2 per repeating / monomeric unit, of the sulfated GAG polysaccharide moieties at a pH in the range of 6.0 to 8.0, in particular at a pH in the range of 7.0 to 7.5, in particular at physiological pH. The inventors of the present invention found that the negative charge of the sulfate group and the thus enabled relatively high solubility and a relatively low viscosity of the polar composition comprising the sulfated GAG polysaccharide moieties allow the formation of microparticles in the emulsion compared to polysaccharide moieties having a relatively low charge per repeating / monomeric unit, in particular a negative net charge of less than -2 per repeating / monomeric unit, in particular between 0 and -1 per repeating / monomeric unit, and thus resulting in a relatively low solubility and a relatively high viscosity of the polar composition. This is the case for example for the disaccharide unit of hyaluronic acid having a negative net charge of -1 per repeating / monomeric unit.

[0040] Furthermore, the inventors of the present invention found that polysaccharide moieties having a relatively low charge per repeating / monomeric unit, in particular hyaluronic acid, show a further reduced solubility and further increased viscosity of the polar composition, if the polysaccharide moieties having a relatively low charge per repeating / monomeric unit have a relatively long polymer chain length, in particular a polymer chain length, in particular a hyaluronic acid polymer chain length, of more than 250 repeating / monomeric units per polysaccharide moiety, in particular of more than 500 repeating / monomeric units per polysaccharide moiety.Merz Aesthetics GmbH 120725P502PCNovember 7, 2025

[0041] Within the context of the present invention, the molecular mass / molecular weight of the sulfated GAG polysaccharide moieties is not particularly limited and may be, for example, in the range of 10 kDa and 5000 kDa, preferably in the range of 100 kDa and 2000 kDa, preferably in the range of 250 kDa and 1500 kDa, preferably in the range of 500 kDa and 1000 kDa. The sulfated GAG polysaccharide moieties may also comprise a mixture of a high molecular weight sulfated GAG polysaccharide moiety having a molecular weight in the range of 1000 kDa and 5000 kDa, preferably in the range of 2000 kDa and 4000 kDa, preferably in the range of 2500 kDa and 3000 kDa, and a low molecular weight sulfated GAG polysaccharide moiety having a molecular weight in the range of 10 kDa and 1000 kDa, preferably in the range of 100 kDa and 750 kDa, preferably in the range of 250 kDa and 500 kDa, in a ratio in the range of, e.g., 10:90 to 90:10.

[0042] Furthermore, the term “comprise”, as used herein, for example in the context of “a microparticle comprising crosslinked sulfated GAG polysaccharide moieties...” or “a method comprising...”, is intended to encompass both the open-ended term “includes” and the closed-ended term “consisting of”.

[0043] In a first aspect, the present invention relates to a method for preparing a microparticle comprising crosslinked sulfated glycosaminoglycan (GAG) polysaccharide moieties, the method comprising the steps of:(a) providing a polar composition comprising a polar solvent and sulfated GAG polysaccharide moieties, wherein the sulfated GAG polysaccharide moieties comprise carboxylic acid groups and nucleophilic groups,(b) providing a non-polar composition comprising a non-polar solvent,(c) mixing the polar composition and the non-polar composition to obtain an emulsion comprising microparticles comprising sulfated GAG polysaccharide moieties, wherein the emulsion is a reverse emulsion,(d) adding a carboxyl-activating agent to the emulsion obtained in step (c) to activate at least some of the carboxylic acid groups,Merz Aesthetics GmbH 120725P502PCNovember 7, 2025(e) allowing reaction of at least some of the activated carboxylic acid groups of the sulfated GAG polysaccharide moieties with at least some of the nucleophilic groups of the sulfated GAG polysaccharide moieties to obtain microparticles comprising crosslinked sulfated GAG polysaccharide moieties, and(f) purifying the microparticles comprising crosslinked sulfated GAG polysaccharide moieties.

[0044] The term “polar composition” means a liquid or a solution that comprises a polar solvent as a component, in particular as the single largest liquid component, in particular as a major liquid component, that is a composition that comprises a polar solvent content by weight of more than 50 wt.%, in particular of at least 60 wt.%, in particular of at least 70 wt.%, in particular of at least 80 wt.%, in particular of at least 90 wt.%, in particular of at least 95 wt.%, based on the total mass of the polar composition. The polar composition and / or the polar solvent may have a Snyder's polarity index in the range of 5.0 to 10.2, in particular in the range of 5.5 to 9.6, in particular in the range of 6.0 to 8.8, in particular in the range of 6.5 to 7.2. Snyder's polarity index is a known classification parameter of solvents and can be calculated as described in L.R. Snyder, Classification of the solvent properties of common liquids, J. Chrom. Sci., 1978, 16, 223-234. The Snyder polarity index P' is well known in the art for a wide variety of solvents and mixtures of solvents and can readily be determined by the skilled person for a given solvent system. In particular, the polar solvent may comprise water. In this regard, the term “water”, as used herein, may be understood in the broadest sense. Preferably, water may be deionized water, distilled water, or tap water. Preferably, water may be deionized water or distilled water. The sulfated GAG polysaccharide moieties and optionally salts and / or at least one polar cargo molecule may be dissolved in the polar composition.

[0045] The polar composition may be an aqueous solution. An aqueous solvent may be understood in the broadest sense as a solvent that comprises a water content by weight of more than 50 wt.%, of at least 60 wt.%, of at least 70 wt.%, of at least 80 wt.%, of at least 90 wt.%, of at least 95 wt.%, or even 100 wt.%, referred to the total mass of the solvent. The polar composition may further comprise one or more components selected from the group consisting of one or more alcohols (in particular one or more C1-C5 alcohols such as, e.g.,Merz Aesthetics GmbH 120725P502PCNovember 7, 2025 methanol, ethanol, n-propanol, isopropanol, n-butanol (1 -butanol), sec-butanol (2-butanol), isobutanol, (2-methyl-propan-1-ol), tert-butanol (2-methylpropan-2-ol), pentan-1 -ol, 2- methylbutan-1-ol, 3-methylbutan-1-ol, 2,2-dimethylpropan-1-ol, pentan-2-ol, 3-methylbutan- 2-ol, pentan-3-ol, and / or 2-methylbutan-2-ol), one or more primary amines (in particular one or more Ci-Cs-amines), one or more carbonic acids (in particular one or more Ci-Cs-carbonic acids such as, e.g., formic acid, acetic acid, propionic acid, butyric acid, valerianic acid, isovalerianic acid), one or more primary or secondary amides (in particular one or more Ci- Cs-amides such as, e.g., formamide), one or more sulfoxides (in particular one or more Ci- Cs-sulfoxides such as, e.g., dimethyl sulfoxide (DMSO)), one or more inorganic or organic cations (in particular one or more inorganic or organic cations of a molecular weight or less than 1000 Da, in particular alkali cations or earth alkali cations, other metal cations, protons, ammonium cations, etc.), one or more inorganic or organic anions (in particular one or more inorganic or organic anions of a molecular weight or less than 1000 Da, in particular chlorine, sulfate, etc.), one or more silicates, and a combination of two or more thereof. In particular, the aqueous solution may comprise, in addition to water, one or more components selected from the aforementioned groups. In particular, the polar composition may comprise water and / or a sulfoxide, in particular a Ci-Cs-sulfoxide, in particular DMSO.

[0046] The polar composition may be an aqueous buffer, in particular a phosphate- buffered saline (PBS) buffer, a Tris buffer, a borate buffer or an acetic acid-buffered buffer. Preferably, the polar composition may be an aqueous buffer having a pH in the range of 5.0 to 9.0, in particular 6.0 to 8.0, in particular 6.1 to 7.9, in particular 6.5 to 7.7, in particular 6.7 to 7.5, in particular 7.0 to 7.4. In particular, the polar composition may be an aqueous buffer having a pH in the range of 6.0 to 8.0.

[0047] Preferably, the polar composition may further comprise at least one polar cargo molecule. The polar cargo molecule may be a hydrophilic cargo molecule. In the context of the present invention, hydrophilicity refers to the ability of a material or compound to absorb, swell, or dissolve in water. Hydrophilicity means that compounds comprising polar functional groups combine with water molecules through electrostatic interaction or hydrogen bonding. By providing at least one polar cargo molecule in the polar composition in step (a), the at leastMerz Aesthetics GmbH 120725P502PCNovember 7, 2025 one polar cargo molecule is encapsulated in the microparticles during step (c). The polar cargo molecule may be selected from the group consisting of drugs, oligonucleotides, peptides, oligopeptides, polypeptides, monosaccharides, disaccharides, polysaccharides, carbohydrates, adjuvants, small molecules and cytokines, and / or may have a weight average molecular weight in the range of 50 g / mol and 10000 g / mol, in particular in the range of 100 g / mol and 7500 g / mol, in particular in the range of 200 g / mol and 5000 g / mol, in particular in the range of 250 g / mol and 2500 g / mol, in particular in the range of 500 g / mol and 1000 g / mol. The polar cargo molecule may be soluble in the polar composition and may form a polar solution. In particular, the polar cargo molecule may be soluble in water. If the polar cargo molecule comprises nucleophilic groups, in particular amine groups and / or hydroxy groups, at least some of the nucleophilic groups may react with at least some of the activated carboxylic acid groups of the sulfated GAG polysaccharide moieties in step (e), thereby forming a covalent linkage, in particular an ester linkage and / or an amide linkage, between the sulfated GAG polysaccharide moieties and the polar cargo molecule. The polar cargo molecule may have a dipole moment. The polar cargo molecule may have a polarity comparable to the polarity of the polar solvent.

[0048] The term “non-polar composition” refers to a liquid or a solution that comprises a non-polar solvent as a component, in particular as the single largest liquid component, in particular as a major liquid component, that is a composition that comprises a non-polar solvent content by weight of more than 50 wt.%, in particular of at least 60 wt.%, in particular of at least 70 wt.%, in particular of at least 80 wt.%, in particular of at least 90 wt.%, in particular of at least 95 wt.%, in particular 100 wt.%, based on the total mass of the non-polar composition. The non-polar composition and / or the non-polar solvent may have a Snyder's polarity index in the range of 0.0 to 4.8, in particular in the range of 0.1 to 4.1 , in particular in the range of 1 .0 to 4.0, in particular in the range of 2.5 to 3.5. As indicated above with respect to the polar composition and / or the polar solvent, the Snyder's polarity index P' is a known classification parameter of solvents which can readily be determined by the skilled person for a given solvent system. The non-polar solvent may be any non-polar solvent. In particular, the non-polar solvent may be a hydrocarbon. The hydrocarbon may be pentane, cyclopentane, hexane, cyclohexane, heptane, iso-octane, petroleum ether, n-butyl chloride, methyl t-butyl ether, xylene, benzene, chlorobenzene, o-dichlorobenzene, ethyl ether, diethylMerz Aesthetics GmbH 120725P502PCNovember 7, 2025 ether, toluene, dichloromethane, ethylene dichloride, n-butyl alcohol, isopropyl alcohol, n- butyl acetate, isobutyl alcohol, methyl isoamyl ketone, n-propyl alcohol, tetrahydrofuran, chloroform, methyl isobutyl ketone, methyl n-propyl ketone, methyl ethyl ketone, 1 ,4-dioxane, a fatty acid or mixtures thereof. In particular, the fatty acid may be oleic acid. In particular, the non-polar solvent may be heptane. The inventors of the present invention found that the purification of the microparticles comprising crosslinked sulfated GAG polysaccharide moieties in the non-polar solvent is particularly improved, if a non-polar solvent having a relatively low boiling point, in particular heptane, is used due to the facilitated removal of residual solvent in vacuo.

[0049] The non-polar composition (and / or the non-polar solvent) and the polar composition (and / or the polar solvent) may be selected such that they are immiscible and form a two- phase system, in particular a two-phase emulsion, in particular a two-phase reverse emulsion, upon mixing. Thus, the non-polar composition may have a miscibility gap with the polar composition. Furthermore, the non-polar solvent may have a miscibility gap with the polar solvent. The term “emulsion” refers to a mixture of two or more liquids in which particles or droplets of a non-polar liquid (e.g. the non-polar solvent) are dispersed in a polar medium (e.g. the polar composition). The mixing of the polar composition and the non-polar composition in step (c) results in the provision of a reverse emulsion. Reverse emulsions are typically mixtures of droplets of polar liquid (e.g. the polar composition comprising the plurality of sulfated GAG polysaccharide moieties) in a non-polar medium (e.g. the non-polar solvent), which is also referred to as “oil-in-water-emulsion”, and comprises a plurality of reverse micelles. If at least one polar cargo molecule is provided in the polar composition in step (a), the polar cargo molecule is encapsulated in the inner part of the reverse micelles in the mixing of step (c).

[0050] Preferably, a surfactant may be provided in the polar composition in step (a) and / or the non-polar composition in step (b). The surfactant may be sodium dodecylsulfate, cetyltrimethylammonium bromide, polyglycerol polyricinoleate (PGPR, E476), Span 20, Span 80, Span 85, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, lecithin, ricinoleic acid ester (CR-310), proteins, in particular -lactoglobulin, whey protein isolatesMerz Aesthetics GmbH 120725P502PCNovember 7, 2025(WPI) and / or casein, lysolecithin, nonylphenoxypolyoxyethylene, lysophosphatidylcholine, polyethylenglycol 400, polyoxyethylene ethers, polyglycol ether surfactants, DMSO, sodium laurate, sodium lauryl sulfate, benzalkonium chloride and mixtures thereof. By provision of a surfactant in the polar composition and / or in the non-polar composition, the stability of the emulsion or the reverse emulsion can be improved. In this regard, the stability of the emulsion or the reverse emulsion refers to the ability of the emulsion or the reverse emulsion to resist changes in its properties over time. Thus, by provision of a surfactant, the kinetic stability of the emulsion or the reverse emulsion can be maintained, which results in a stable size of the droplets over time.

[0051] As already indicated above, the term “sulfated glycosaminoglycan (GAG) polysaccharide moieties” is further specified as structures comprising carboxylic acid groups or salts thereof and nucleophilic groups. The nucleophilic groups may be hydroxy groups. If the sulfated GAG polysaccharide moiety comprises amine groups and / or is a derivative of a sulfated GAG polysaccharide moiety comprising amine groups, the nucleophilic groups may be hydroxy groups and / or amine groups. If the sulfated GAG polysaccharide moieties comprise amine groups, at least some of the activated carboxylic acid groups of the sulfated GAG polysaccharide moieties may react with at least some of the amine groups of the sulfated GAG polysaccharide moieties, wherein the reaction of step (e) results in an amide linkage. Preferably, the sulfated GAG polysaccharide moieties as specified according to the invention may be selected from the group consisting of chondroitin sulfate and / or derivatives thereof, dermatan sulfate, heparin, heparan sulfate and mixtures thereof. The chondroitin sulfate may be selected from the group consisting of chondroitin-4-sulfate, chondroitin-6- sulfate, chondroitin-2, 6-sulfate, chondroitin-4, 6-sulfate and mixtures thereof. The derivative of chondroitin sulfate may be polysulfated chondroitin sulfate and / or a chondroitin sulfate comprising at least one amine group.

[0052] The carboxyl-activating agent may be a crosslinking agent, in particular a zerolength crosslinker. The carboxyl-activating agent may be selected from the group consisting of triazine-based carboxyl-activating agents, in particular 4-(4,6-dimethoxy-1 , 3, 5-triazin-2-yl)- 4-methylmorpholinium (DMTMM) or a salt thereof, carbodiimide-based carboxyl-activatingMerz Aesthetics GmbH 120725P502PCNovember 7, 2025 agent, in particular 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-N'- dicyclohexylcarbodiimide (DCC), diiosopropylcarbodiimide (DIC), or salts thereof, N- hydroxysuccinimide (NHS), glycidyl ethers, maleimides and alkoxides. In particular, the carboxy l-activati ng agent may be a triazine-based carboxyl-activating agent, in particular DMTMM or a salt thereof. In particular, BDDE and / or divinylsulfone (DVS) is / are not used as crosslinking agents for covalent binding. In particular, a polyethylene glycol diglycidyl ether (PEGDE) crosslinker is not used for covalent binding.

[0053] In the context of the present invention, a carboxyl-activating agent is understood as a compound that effects activation of carboxylic acid groups of the sulfated GAG polysaccharide moieties and thus allows subsequent reaction of the activated carboxylic acid groups with nucleophilic groups of the sulfated GAG polysaccharide moieties thereby forming covalent bonds, in particular ester bonds. The carboxyl-activating agent is typically not covalently included in the crosslinked material (so-called zero-length crosslinker) resulting in an improved safety profile of the crosslinked material due to the absence of xenobiotic linker structures. Thus, the carboxyl-activating agent may be removed from the crosslinked material by any means such as, e.g., washing, filtration, etc. Furthermore, the formed covalent bonds, in particular the formed ester bonds, can be easily degraded enzymatically, in particular by hydrolysis, by administration of an enzyme, in particular hyaluronidase, which allows a correction if necessary or desired.

[0054] The term “triazine-based carboxyl-activating agent”, as used herein, refers to a compound based on a triazine core structure that effects reaction of carboxylic acid groups with nucleophilic groups, in particular hydroxy groups, thereby forming (intermolecular) bonds, in particular (intermolecular) ester bonds. In this regard, the triazine-based carboxyl- activating agent may have any structure that comprises a triazine core that is suitable for activating at least some of the carboxylic acid groups of the sulfated GAG polysaccharide moieties and allowing reaction of at least some of the activated carboxylic acid groups with at least some of the nucleophilic groups of the sulfated GAG polysaccharide moieties thereby crosslinking the sulfated GAG polysaccharide moieties covalently with each other. As an example, the triazine-based carboxyl-activating agent may have the structure:Merz Aesthetics GmbH 120725P502PCNovember 7, 2025wherein:R1, R2and R3are independently from each other any residue, such as, e.g., each independently a residue selected from the group consisting of hydrogen, deuterium, Ce-Cw- aryl, C2-Cw-heteroaryl, linear or branched Ci-Cw-(cyclo)alkyl, linear or branched C1-C10- (cyclo)alkoxy, linear or branched Ci-Cw-hetero(cyclo)alkyl, linear or branched C2-C10- (cyclo)alkenyl, C2-Cwhetero(cyclo)alkenyl, wherein each of the aforementioned residues is optionally substituted with one or more residues selected from the group consisting of halogen, Ce-C -aryl, C2-Cw-heteroaryl, linear or branched Ci-Cio-(cyclo)alkyl, linear or branched Ci-Ciohetero(cyclo)alkyl, linear or branched Ci-Cio-(cyclo)alkoxy, linear or branched C2-Cio-(cyclo)alkenyl, C2-Cw-hetero(cyclo)alkenyl, linear or branched C2- Cw(cyclo)alkinyl, and C2-Cio-hetero(cyclo)alkenyl, or a salt thereof.

[0055] Preferably, at least one of the residues R1, R2and R3may be an optionally substituted morpholinium residue. Preferably, at least one of the residues R1, R2and R3may be a Ci-C4-alkyl-morpholinium residue. Preferably, at least one of the residues R1, R2and R3may be a methylmorpholinium residue. Preferably, at least one of the residues R1, R2and R3may be an optionally substituted linear or branched Ci-Cio-(cyclo)alkoxy residue. Preferably, at least one of the residues R1, R2and R3may be an optionally substituted linear or branched Ci-C4-alkoxy residue. Preferably, at least one of the residues R1, R2and R3may be an optionally substituted linear or branched methoxy residue.

[0056] The triazine-based carboxyl-activating agent may have a molecular weight of not more than 1500 g / mol, in particular of not more than 1000 g / mol, in particular of not more than 500 g / mol, in particular of not more than 400 g / mol. Preferably, the triazine-based carboxyl-activating agent may be selected from the group consisting of 4-(4,6-dimethoxy- 1 ,3,5-triazin-2-yl)-4-methylmorpholinium (DMTMM) or a salt thereof, and / or 2-chloro-4,6,- dimethoxy-1 ,3,5-triazine or a salt thereof, and combinations thereof. Preferably, the triazine-Merz Aesthetics GmbH 120725P502PCNovember 7, 2025 based carboxyl-activating agent may be 4-(4,6-dimethoxy-1 ,3,5-triazin-2-yl)-4- methylmorpholinium (DMTMM) or a salt thereof. A salt of DMTMM is preferably a salt, wherein the counter-ion is an anion that is cosmetically and / or pharmaceutically acceptable such as, e.g., chloride, acetate, bicarbonate (hydrogen carbonate), or a mixture of two or more anions. The CAS No. of the chloride salt of DMTMM is 3945-69-5.

[0057] In particular, the salt of DMTMM may be a tetrafluoroborate salt. By using a tetrafluoroborate salt of DMTMM, the solubility of the DMTMM can be further increased. DMTMM is considered as having a comparably low and essentially negligible toxicity, is not cancerogenic, not mutagenic, and not teratogenic / reprotoxic in the generally used amounts. Thus, it is particularly well usable for preparing a soft-tissue filler such as a dermal or connective tissue filler.

[0058] When using DMTMM or a salt thereof as carboxyl-activating agent, 4- methylmorpholine (NMM) and / or 4, 6-dimethoxy-1 ,3-5-triazine-2-ol (DMT) may be formed as degradation product(s). For example, the chloride salt of DMTMM may be used as carboxyl- activating agent:

[0059] The emulsion, in particular the reverse emulsion, can be obtained by mixing the polar composition as specified according to the present invention and the non-polar composition as specified according to the invention in a mixer, a paddle mixer, a Henschel mixer, a homogenizer, a colloid mill, a propeller stirrer, an extruder, an ultrasonic emulsifier, or a vacuum emulsifier.

[0060] Preferably, the mixing may be performed at a volume ratio of the non-polar composition to the polar composition of 95:5 to 55:45, in particular 90: 10 to 60:40, in particular 85:15 to 65:35, in particular 80:20 to 70:30. In order to provide the emulsion, in particular theMerz Aesthetics GmbH 120725P502PCNovember 7, 2025 reverse emulsion, in step (c), a volume excess of the non-polar composition to the polar composition may be provided. The inventors of the present invention found that the size of the obtained microparticles comprising sulfated GAG polysaccharide moieties, can be controlled by means of the volume excess of the non-polar composition to the polar composition. The inventors of the present invention found that with an increased excess of the non-polar composition to the polar composition in the mixing step (c), the obtained microparticles have a smaller average equivalent spherical diameter D50.

[0061] In step (c), the mixing may be performed under stirring. The mixing of the polar composition and the non-polar composition under stirring results in the size-controllable preparation of microparticles, in particular spherical microparticles, in particular microspheres. The stirring may be performed at a stirring speed of between 500 rpm and 1500 rpm, in particular between 750 rpm and 1250 rpm, in particular between 800 rpm and 1100 rpm, in particular between 900 rpm and 1000 rpm. The stirring speed (in rpm), as used herein, is based on a 1 -liter batch size. For lager batch sizes, the stirring speed is adjusted according to the used volume and / or geometry of the vessel. In this regard, the skilled person knows how to adjust the stirring speed in relation to the used volume and / or geometry of the vessel (e.g. a larger volume requires an increased stirring speed). Preferably, the mixing intensity, in particular by stirring, may be adjusted in the step of mixing according to step (c). Preferably, the mixing intensity, in particular the stirring speed, may be increased during step (c). The stirring speed may be adjusted, in particular increased, to a stirring speed where the formed emulsion has a droplet size, in particular a microparticle size, which is desired. The stirring speed can be adjusted by means of a stirrer.

[0062] In step (e), the reaction is commonly carried out under conditions of a temperature (Tx) of at least 15 °C, in particular at least 25 °C, in particular at least 30 °C, in particular at least 40 °C, wherein the temperature (Tx) and the reaction time (tx) preferably satisfy the equation: (Tx)2tx= y, with y being in the range of from 0.5 x 104(°C)2h to 3 x 105(°C)2h, in particular in the range of 1 x 104(°C)2h to 2 x 105(°C)2h. The reaction temperature is preferably in the range of 20 °C to 80 °C, in particular in the range of 30 °C to 60 °C, inMerz Aesthetics GmbH 120725P502PCNovember 7, 2025 particular in the range of 40 °C to 50 °C. Furthermore, the reaction pH may be in the range of5.0 to 9.0, in particular in the range of 6.0 to 8.0, in particular in the range of 7.0 to 7.5.

[0063] The concentration of the sulfated GAG polysaccharide moieties in the polar composition (i.e. in the polar composition provided in step (a)) is typically between 50 mg / ml and 150 mg / ml, in particular between 75 mg / ml and 125 mg / ml, in particular about 100 mg / ml. The degree of crosslinking, expressed as the molar ratio of carboxyl-activating agent (e.g., DMTMM) added to the emulsion in step (d) and subjected to crosslinking in step (e) to the carboxylic acid groups of the sulfated GAG polysaccharide moieties present in the polar composition provided in step (a), arranged to microparticles in steps (c) and (d) and subjected to crosslinking in step (e) may be in the range of 1 :100 to 100:1 , in particular in the range of 1 : 10 to 10: 1 , in particular in the range of 1 :5 to 5: 1 , in particular in the range of 1 :2 to 2: 1 . The inventors of the present invention found that a relatively high molar ratio of carboxyl-activating agent to the carboxylic acid groups of the sulfated GAG polysaccharide moieties results in microparticles having a relatively high degree of crosslinked sulfated GAG polysaccharide moieties. Furthermore, a relatively low molar ratio of carboxyl-activating agent to the carboxylic acid groups of the sulfated GAG polysaccharide moieties results in microparticles having a relatively low degree of crosslinked sulfated GAG polysaccharide moieties. The inventors of the present invention further found that the degree of crosslinked sulfated GAG polysaccharide moieties of the microparticles affects the enzymatic degradability of the microparticles. In this regard, microparticles having a relatively high degree of crosslinked sulfated GAG polysaccharide moieties show a relatively high resistance to enzymatic degradation.

[0064] The carboxyl-activating agent, in particular the triazine-based carboxyl-activating agent, may be used in any content suitable for crosslinking at least some carboxylic acid groups of the sulfated GAG polysaccharide moieties with at least some of the nucleophilic groups of the sulfated GAG polysaccharide moieties to form covalent bonds, in particular ester bonds, thereby crosslinking the sulfated GAG polysaccharide moieties covalently with each other. In this regard, the carboxyl-activating agent, in particular the triazine-based carboxyl-activating agent, typically activates the carboxylic acid groups of the sulfated GAGMerz Aesthetics GmbH 120725P502PCNovember 7, 2025 polysaccharide moieties. Thus, the content of the carboxyl-activating agent, in particular the triazine-based carboxyl-activating agent, may be reasonably defined as molar equivalents (eq.) related to the carboxylic acid groups of the sulfated GAG polysaccharide moieties. In this context, the term “carboxylic acid groups” also includes salts thereof. The molar equivalents of the carboxyl-activating agent is defined as molar ratio of carboxyl-activating agent to carboxylic acid groups of the sulfated GAG polysaccharide moieties. For this purpose, in the context of chondroitin sulfate as an example, it may be assumed that each chondroitin sulfate monomeric / repeating unit (having a molecular weight (MW) of approximately 463 g / mol) bears one carboxylic acid group. Similar assumptions can be made for dermatan sulfate (having a MW of approximately 475 g / mol) and heparan sulfate (MW of approximately 637 g / mol). In the context of heparin, it may be assumed that each heparin monomeric / repeating unit (having a molecular weight (MW) of approximately 1040 g / mol) bears two carboxylic acid groups.

[0065] After crosslinking, the microparticles comprising crosslinked sulfated GAG polysaccharide moieties obtained in step (e) are purified in step (f). The purifying may be performed by dialysis, in particular by dialysis against a phosphate buffer, by washing and / or by filtration.

[0066] The dialysis may be carried out at elevated temperatures of at least 10 °C, in particular at least 20 °C, in particular at least 30 °C, in particular at least 40 °C, in particular at least 50 °C. The upper limit is determined by the tendency of the microparticles comprising crosslinked sulfated GAG polysaccharide moieties to degrade at excessively high temperatures. The person skilled in the art will be readily select appropriate temperatures to limit degradation to an acceptable level. Although not intended to be limiting, the upper temperature limit may be 80 °C, in particular 70 °C, in particular 60 °C. Thus, exemplary suitable temperature ranges are 20 °C to 80 °C, 30 °C to 70 °C, or 40 °C to 60 °C.

[0067] The dialysis buffer used in dialysis commonly has a pH of about 6.2 to 7.9, in particular a pH of about 6.5 to 7.8, in particular a pH of about 6.8 to 7.4. The buffer used may be a phosphate buffer, and in particular a phosphate buffer with a pH within the rangesMerz Aesthetics GmbH 120725P502PCNovember 7, 2025 mentioned above. Suitable phosphate buffers preferably have a phosphate concentration of about 0.01 mM to about 120 mM, in particular about 0.1 mM to about 100 mM, in particular about 1.0 mM to about 75 mM. Within the context of the present invention, the phosphate concentration refers to the total concentration of all phosphate species (i.e. PO43HPO42', H2PO4 H3PO4) present in the dialysis buffer. It is calculated as the total amount (in moles) of phosphate salts, and optionally phosphoric acid, added to the dialysis buffer divided by the volume of the dialysis buffer (in liters) and expressed in mM or M. The dialysis may be carried out for 6 h to 96 h, in particular for 20 h to 50 h. The volume of the dialysis buffer is commonly between 50-fold to 200-fold the volume of the sample to be dialyzed. Other suitable conditions for dialysis will be readily apparent to those skilled in the art.

[0068] The filtration may be crossflow filtration, dead-end filtration, dynamic filtration or a combination thereof. In the context of filtration, the filter may have any pore size suitable for purifying the microparticles comprising crosslinked sulfated GAG polysaccharide moieties, in particular withholding the microparticles comprising crosslinked sulfated GAG polysaccharide moieties and allowing the passage of reactants and optionally of non-reacted sulfated GAG polysaccharide moieties. Preferably, the pore size may be in the range of 5 nm to 2 pm, in particular of 50 nm to 1 pm, in particular of 100 nm to 500 nm. The filter material may be a ceramic, a metal, a polymer or a combination thereof.

[0069] In a second aspect, the present invention relates to a microparticle obtainable by the method according to the invention. The microparticle obtainable by the method according to the invention may have the same properties of the microparticle according to the following third aspect of the invention.

[0070] In a third aspect, the present invention relates to a microparticle comprising sulfated GAG polysaccharide moieties, wherein the sulfated GAG polysaccharide moieties are covalently crosslinked with each other by ester bonds between at least some of the carboxylic acid groups of the sulfated GAG polysaccharide moieties and at least some of the nucleophilic groups of the sulfated GAG polysaccharide moieties, in particular without an interconnecting linker structure.Merz Aesthetics GmbH 120725P502PCNovember 7, 2025

[0071] The microparticles may have an average equivalent spherical diameter D50 in the range of 8 pm and 100 pm, in particular 10 pm and 90 pm, in particular 12 pm and 80 pm, in particular 15 pm and 70 pm, in particular 20 pm and 60 pm, in particular 25 pm and 45 pm. In particular, the average equivalent spherical diameter D50 may be in the range of 25 pm and 45 pm. The average equivalent spherical diameter D50 of the microparticles can be measured by any convenient means, in particular by laser diffraction analysis, optical imaging (e.g. microscopy, static image analysis, dynamic image analysis) or light scattering. The microparticles may also be characterized by the polydispersity index (PDI). Furthermore, the microparticles with the optionally encapsulated polar cargo molecule may be characterized by the kinetics of the release rate of the polar cargo molecule.

[0072] In a fourth aspect, the present invention relates to the microparticle according to the invention for use as a medicament, in particular for the treatment of degenerative joint diseases, in particular osteoarthritis, or connective tissue diseases, in particular rheumatoid arthritis. The treatment of degenerative joint diseases, in particular osteoarthritis, and / or connective tissue diseases, in particular rheumatoid arthritis, may comprise the steps of administering to a patient suffering from a degenerative joint disease, in particular from osteoarthritis, and / or from a connective tissue disease, in particular rheumatoid arthritis, a therapeutically effective amount of the microparticles. The microparticles, in particular the microparticles with the encapsulated polar cargo molecules, may be used as a drug delivery system. The drug delivery system comprises a microparticle as described above and at least one polar cargo molecule, in particular a drug, encapsulated in the microparticles. In particular, the drug delivery system is for use as a medicament.

[0073] In a fifth aspect, the present invention relates to a hydrogel comprising the microparticle(s) according to the invention, an aqueous solution, and optionally at least one polar cargo molecule, wherein the at least one polar cargo molecule is encapsulated in the microparticle(s).Merz Aesthetics GmbH 120725P502PCNovember 7, 2025

[0074] Furthermore, the hydrogel may comprise additional substances such as cells, including stem cells, and adipocytes, fat, lipids, growth factors, cytokines, drugs, and bioactive. More specifically, the hydrogel may comprise local anesthetic agents, polyalcohols, vitamins, alkali metal and alkaline earth metal salts, metals, antioxidants, amino acids, and ceramic particles.

[0075] Within the context of the present invention, the addition of a local anesthetic is particularly desirable in view of its ability to mitigate pain upon injection. Exemplary local anesthetic agents include, but are not limited to, ambucaine, amolanone, amylocaine, benoxinate, benzocaine, betoxycaine, biphenamine, bupivacaine, butacaine, butamben, butanilicaine, butethamine, butoxycaine, carticaine, chloroprocaine, cocaethylene, cocaine, cyclomethycaine, dibucaine, dimethisoquin, dimethocaine, diperodon, dicyclomine, ecgonidine, ecgonine, ethyl chloride, etidocaine, beta-eucaine, euprocin, fenalcomine, formocaine, hexylcaine, hydroxytetracaine, isobutyl p-aminobenzoate, leucinocaine mesylate, levoxadrol, lidocaine, mepivacaine, meprylcaine, metabutoxycaine, methyl chloride, myrtecaine, naepaine, octocaine, orthocaine, oxethazaine, parethoxycaine, phenacaine, phenol, piperocaine, piridocaine, polidocanol, pramoxine, prilocaine, procaine, propanocaine, proparacaine, propipocaine, propoxycaine, pseudococaine, pyrrocaine, ropivacaine, salicyl alcohol, tetracaine, tolycaine, trimecaine, zolamine, and salts thereof.

[0076] Preferably, the anesthetic agent is lidocaine, such as in the form of lidocaine HCI. The hydrogel may have a lidocaine concentration of, for example, 0.05 wt.% to 8.0 wt.%, 0.1 wt.% to 4.0 wt.%, 0.2 wt.% to 3.0 wt.%, 0.3 wt.% to 2.0 wt.%, or 0.4 wt.% to 1 .0 wt.%.

[0077] Suitable polyols for use herein include, but are not limited to, glycerol, mannitol, sorbitol, propylene glycol, erythritol, xylitol, maltitol, and lactitol. Particularly suitable for use herein is mannitol and glycerol. Further, the polyol is preferably glycol, optionally in combination with one or more of the aforementioned polyol compounds, in particular mannitol. Suitable vitamins include vitamin C, vitamin E and vitamins of the B group, i.e. one or more of B1 , B2, B3, B5, B6, B7, B9 and B12 vitamins. The vitamins may be present to stimulate and maintain cellular metabolism and, thus, to promote collagen production. ParticularlyMerz Aesthetics GmbH 120725P502PCNovember 7, 2025 preferred for use here is vitamin C, vitamin E and vitamin B6. The ceramic particles are preferably hydroxyapatite particles, e.g., calcium hydroxyl apatite (CaHA) particles.

[0078] Furthermore, a dermal filler comprising the hydrogel according to the invention may be provided. Said dermal filler may be provided in a kit comprising said dermal filler according to the invention, and optionally instructions for use.

[0079] The kit may contain at least one pre-filled syringe containing the hydrogel according to the fifth aspect of the present invention, and optionally instructions for use. The kit is suitable for use in various cosmetic (aesthetic) treatments.

[0080] In a sixth aspect, the present invention relates to the use of the microparticles according to the present invention or the hydrogel according to the present invention for cosmetic applications.

[0081] Exemplary cosmetic applications in accordance with the present invention include, but are not limited to, augmenting or filling of wrinkles and lines of the skin, in particular of facial lines and facial wrinkles (e.g., glabellar lines, nasolabial folds, chin folds, marionette lines, buccal commissures, peri-oral wrinkles, peri-lip wrinkles, and crow's feet). Other exemplary cosmetic applications include treating the perioral region, facial asymmetries, jawlines, chin, cheeks, nose, lips, and infraorbital region as well as improving skin hydration and / or skin texture.

[0082] Furthermore, a method for replacing or filling of a biological tissue or increasing the volume of a biological tissue for cosmetic purposes, in particular for cosmetic treatments of skin lines and wrinkles, comprising administering to a subject in need thereof an effective amount of the dermal filler comprising the hydrogel according to the invention may be provided.

[0083] The term "effective amount", as used herein, is generally intended to refer to the amount of the dermal filler comprising the hydrogel according to the invention sufficient toMerz Aesthetics GmbH 120725P502PCNovember 7, 2025 effect beneficial or desired cosmetic (aesthetic) results. A "subject" in the sense of the present invention is any individual or patient, usually a human, in need of a treatment of a particular condition.

[0084] The dermal filler comprising the hydrogel according to the invention is generally administered by injection, more specifically by subcutaneous or intradermal injection, using techniques known in the art such as the serial puncture technique. In particular, the dermal filler comprising the hydrogel according to the invention may be injected into the dermis and / or the subcutis, preferably into the deep dermis and / or upper subcutis.

[0085] The following non-limiting example further illustrate the present invention.EXAMPLEFormation of microparticles and encapsulation of hydrophilic / polar compounds depending on the volume ratio of the non-polar composition to the polar composition

[0086] A polar composition of 100 mg / mL chondroitin sulfate polysaccharide (Chondroitin- 4-sulfate, MW: 109.751 g / mol, corresponding to approximately 236 chondroitin sulfate monosaccharide units per polysaccharide) containing 10% (w / v) sodium dodecyl sulfate (SDS) as surfactant is prepared in PBS (pH 7.4). In order to show the encapsulation of hydrophilic / polar cargo molecules, 50 pL of Rhodamine B (100 mmol) is dissolved in the polar composition. As the non-polar composition, heptane is used. The polar composition and the non-polar composition are mixed in different volume ratios of the non-polar composition to the polar composition of 70:30 (v / v), 75:25 (v / v) or 80:20 (v / v) in 50 mL round bottom flasks by stirring the mixture at 900 rpm, respectively. In all three samples, an emulsion with chondroitin sulfate microparticles was obtained. Subsequently, 0.5 eq. of DMTMM relative to the COOH groups of chondroitin sulfate are dissolved in the emulsions, respectively (e.g. 374 mg of DMTMM in the 70:30 (v / v) emulsion). The crosslinking reaction takes place at room temperature for 17 hours. After crosslinking, the crosslinked hydrogel is purified with dialysis against PBS buffer and analyzed. The average equivalent spherical diameter D50 of the crosslinked microspheres (microparticles) are analyzed in Table 1.Merz Aesthetics GmbH 120725P502PCNovember 7, 2025Table 1.

[0087] As can be seen from Table 1 , microparticles comprising crosslinked chondroitin sulfate polysaccharide moieties with an average equivalent spherical diameter D50 in the range of 11 to 16 pm are obtained. In this regard, an increase in the ratio of the non-polar composition to the polar composition results in a decrease of the equivalent spherical diameter D50.

[0088] Furthermore, the chondroitin sulfate microspheres were analyzed by fluorescence microscopy after crosslinking using DMTMM. The encapsulation of the fluorescent dye rhodamine B within the crosslinked microparticles was verified. Furthermore, no change of color of the dialysis solution is observed after 72 hours, indicating that no involuntary slow release of the encapsulated rhodamine B from the crosslinked microparticles occurs and indicating that the crosslinked microparticles are stable.

Claims

1. Merz Aesthetics GmbH 120725P502PCNovember 7, 2025CLAIMS1. A method for preparing microparticles comprising crosslinked sulfated glycosaminoglycan (GAG) polysaccharide moieties, the method comprising the steps of:(a) providing a polar composition comprising a polar solvent and sulfated GAG polysaccharide moieties, wherein the sulfated GAG polysaccharide moieties comprise carboxylic acid groups and nucleophilic groups,(b) providing a non-polar composition comprising a non-polar solvent,(c) mixing the polar composition and the non-polar composition to obtain an emulsion comprising microparticles comprising sulfated GAG polysaccharide moieties, wherein the emulsion is a reverse emulsion,(d) adding a carboxyl-activating agent to the emulsion obtained in step (c) to activate at least some the carboxylic acid groups,(e) allowing reaction of at least some of the activated carboxylic acid groups of the sulfated GAG polysaccharide moieties with at least some of the nucleophilic groups of the sulfated GAG polysaccharide moieties to obtain microparticles comprising crosslinked sulfated GAG polysaccharide moieties, and(f) purifying the microparticles comprising crosslinked sulfated GAG polysaccharide moieties.

2. The method of claim 1 , wherein the sulfated GAG polysaccharide moieties are selected from the group consisting of chondroitin sulfate and / or derivates thereof, dermatan sulfate, heparin, heparan sulfate and mixtures thereof.

3. The method of claim 1 or 2, wherein the carboxyl-activating agent is selected from the group consisting of triazine-based carboxyl-activating agents, in particular 4-(4,6-dimethoxy-1 ,3,5-triazin-2-yl)-4-methylmorpholinium (DMTMM) ora salt thereof, carbodiimide-based carboxyl-activating agents, in particular 1 -ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC), N-N'-dicyclohexylcarbodiimide (DCC),Merz Aesthetics GmbH 120725P502PCNovember 7, 2025 diiosopropylcarbodiimide (DIC), or salts thereof, N-hydroxysuccinimide (NHS), glycidyl ethers, maleimides and alkoxides.

4. The method of any one of claims 1 to 3, wherein the polar composition further comprises at least one polar cargo molecule, wherein the at least one polar cargo molecule is encapsulated in the microparticles during step (c).

5. The method of claim 4, wherein the at least one polar cargo molecule is selected from the group consisting of drugs, oligonucleotides, peptides, oligopeptides, polypeptides, monosaccharides, disaccharides, polysaccharides, carbohydrates, adjuvants, small molecules and cytokines, and / or wherein the at least one polar cargo molecule has a weight average molecular weight in the range of 50 g / mol and 10000 g / mol.

6. The method of any one of claims 1 to 5, wherein in step (c) the mixing is performed at a volume ratio of the non-polar composition to the polar composition of 95:5 to 55:45, and / or wherein in step (c) the mixing is performed under stirring, in particular at a stirring speed of between 500 rpm and 1500 rpm.

7. The method of any one of claims 1 to 6, wherein the nucleophilic group is a hydroxy group or an amine group, in particular a hydroxy group, preferably the nucleophilic group is a hydroxy group and the reaction of step (e) results in an ester linkage.

8. The method of any one of claims 1 to 7, wherein in step (d) the molar ratio of the carboxy l-activati ng agent to the carboxylic acid groups of the sulfated GAG polysaccharide moieties is in the range of 1 :100 and 100:1.

9. The method of any one of claims 1 to 8, wherein the purifying in step (f) is performed by dialysis, in particular by dialysis against a phosphate buffer, by washing, and / or by filtration.

10. A microparticle obtainable by a method of any one of claims 1 to 9.

11. A microparticle comprising sulfated glycosaminoglycan (GAG) polysaccharide moieties, wherein the sulfated GAG polysaccharide moieties are covalently crosslinked with each other by ester bonds between at least some of the carboxylicMerz Aesthetics GmbH 120725P502PCNovember 7, 2025 acid groups of the sulfated GAG polysaccharide moieties and at least some of the hydroxy groups of the sulfated GAG polysaccharide moieties.

12. The microparticle of claim 11 , wherein the average equivalent spherical diameter D50 is in the range of between 8 pm and 100 pm, as measured by light scattering.

13. A microparticle of any one of claims 10 to 12 for use as a medicament.

14. A hydrogel comprising a microparticle of any one of claims 10 to 12 and an aqueous solution.

15. Use of the microparticle of any one of claims 10 to 12 or the hydrogel according to claim 14 for cosmetic applications.

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