Granular hydrogel combinations for wound healing

Abstract

A granular hydrogel composition, preferably for the treatment of wounds, comprising a mixture of at least a plurality of first hydrogel microspheres, wherein the first hydrogel microspheres are formed of a first hydrogel scaffold material, and wherein the first hydrogel microspheres further comprise at least one growth factor compound, and at least a plurality of second hydrogel microspheres, wherein the second hydrogel microspheres are formed of a second hydrogel scaffold material, and wherein the second hydrogel microspheres further comprise at least a capturing compound for capturing a proinflammatory compound, wherein the capturing compound is bound to the second hydrogel scaffold material, and optionally a liquid aqueous phase.

Description

[0001] TITLE

[0002] GRANULAR HYDROGEL COMBINATIONS FOR WOUND HEALING

[0003] TECHNICAL FIELD

[0004] The present invention relates to granular hydrogel compositions and their use in the treatment of wounds, especially in diabetic patients.

[0005] PRIOR ART

[0006] A major challenge for the regeneration of chronic wounds is an underlying dysregulation of signaling molecules, including inflammatory cytokines and growth factors.

[0007] To address this, solutions have been proposed in the state of the art which use granular biomaterials composed of microgels, to enable the delivery of biomolecules such as growth factors to the wound and thereby enhance the healing process.

[0008] Heparin Microislands in Microporous Annealed Particle Scaffolds for Accelerated Diabetic Wound Healing. L Pruett, C. Jenkins, N. Singh, K. Catallo, and D. Griffin; Adv. Funct. Mater. 2021, 31, 2104337 discloses a granular hydrogel composition for the treatment of wounds in diabetic patients, comprising a mixture of hydrogel microspheres using 4-arm poly(ethylene glycol) maleimide (PEG-MAL) as the scaffold where a part of the microspheres include heparin and another part of the microspheres is devoid of heparin. The authors report that the heterogeneity in microsphere composition and not simply heparin content is beneficial to increased cell ingrowth in in vitro assays.

[0009] Activating an adaptive immune response from a hydrogel scaffold imparts regenerative wound healing. D. Griffin, M. Archang, C. Kuan, W Weaver, J. Weinstein, A. Feng, A. Ruccia, E. Sideris, . Ragkousis, J. Koh, M. Plikus, D Di Carlo, T. Segura and P. Scumpia; Nature Materials, Vol. 20 April 2021: 560-569 discloses microporous annealed particle (MAP) scaffolds of microgels crosslinked via d-peptides (d-MAP). The authors found that the use of d-peptides enhanced tissue regeneration in skin tissue.

[0010] CN107007881 B discloses a microgel capable of releasing proteins such as growth factors absorbed in the microgel via the degradation rate of the microgel, whose rate can be tuned by the degree of cross-linking of the microgel.

[0011] US2021100927A1 discloses a wound dressing comprising a structural material such as a hydrogel formed into a dressing, and wherein at least one immunomodulatory agent chosen among growth factors is associated with the dressing.

[0012] Programmable Release of Multiple Protein Drugs from Aptamer-Functionalized Hydrogels via Nucleic Acid Hybridization; M. Battig, B. Soontornworajit, and Y. Wang; J. Am. Chem. Soc. 2012, 134, 30, 12410-12413 discloses aptamer-functionalized hydrogels with adjustable release rates at predetermined time points using complementary sequences (CSs) as biomolecular triggers. The hydrogels can be engineered to release growth factors such as VEGF or PDGF-BB.

[0013] While the above provided solutions describe the use of hydrogels as a means to release growth factors in wounds, they do not enable to independently and simultaneously control the concentration of other endogenous factors such as inflammatory cytokines.

[0014] On the other hand, solutions have been proposed in the state of the art which use antibodies to reduce the concentration of inflammatory signal molecules from peripheral blood as in a dialysis process or via an implant placed into blood flow.

[0015] Extracorporeal removal of proinflammatory cytokines by specific absorption onto microspheres; C Weber, D Falkenhagen; ASAIO J. 1996 Sep-Oct;42(5):M908-11; discloses the use of covalently linked polyclonal antibodies against IL-1 beta and TNF alpha onto paramagnetic microspheres in order to remove IL-1 beta and TNF alpha from the bloodstream such as to influence the systemic inflammatory response syndrome and enhance clinical outcome of sepsis.

[0016] Further, non-specific heparin binding in a bulk hydrogel to scavenge interleukins was predicted to be useful in a wound dressing material:

[0017] Glycosaminoglycan-based hydrogels capture inflammatory chemokines and rescue defective wound healing in mice, Lohman et al., Science Translational Medicine, 19 Apr 2017, Vol 9, Issue 386 discloses hydrogels composed of star-shaped polyethylene glycol (starPEG) and glycosaminoglycans (GAGs) were customized to suppress persistent proinflammatory chemokine gradients in chronic wounds. Heparin, carrying the highest anionic charge density of all known biopolymers, was chosen as a generically chemokine- scavenging component in the hydrogel and was found to scavenge interleukins and are predicted to be useful as wound dressing materials.

[0018] Pioggali-Jou et al.: "Growth factor-loaded sulfated microislands in granular hydrogels promote bJISCs migration and chondrogenic differentiation", Acta Biomineralia, Vol. 166, 6 April 2023, pages 69-84, (XP087346519) discloses a granular hydrogel composition, preferably for the treatment of cartilage wounds, comprising a mixture of a plurality of first hydrogel microspheres comprising at least one growth factor compound, and a plurality of second hydrogel microspheres. However, the second hydrogel microspheres do not comprise a bound compound for capturing a proinflammatory compound. The granular hydrogel is meant to recruit chondrocytes from the surrounding cartilage, and favor their ingress.

[0019] Sun L T et al.: "Design principles for cytokine-neutralizing gels: Cross-linking effects", Acta Biomineralia, Vol.. 6, no. 12, 1 December 2010, pages 4708-4715 (XP027423395) discloses hydrogels for wound treatment, wherein said hydrogels comprise antibodies to neutralize pro-inflammatory interleukins. However, this document does not relate to granular hydrogel compositions or granular hydrogel compositions comprising growth factors.

[0020] Us 2019 / 151497 A1 discloses a granular hydrogel composition comprising a mixture of a first hydrogel microspheres, and a second hydrogel microspheres. The microgel particles can be loaded with a plethora of compounds such as growth factors and antibodies, this combination is not taught in and of itself and is not taught in the context of including such a combination in separate microgel particles.

[0021] Therefore, there exists a need to provide hydrogels for wound dressing, in which regeneration of injured tissue can be promoted via the efficient modulation of both inflammatory signals and proliferative signals. SUMMARY OF THE INVENTION

[0022] The present invention provides a granular hydrogel composition for the treatment of wounds in which regeneration of injured tissue can be promoted via the efficient and independent modulation of both inflammatory signals and proliferative signals in the wound, because the granular hydrogel composition is formed by two sub-populations of microgel spheres, where one population is capable of promoting the regeneration and proliferation of cells in the wound and where the other population is capable of reducing inflammation. The microgel spheres can be mixed and matched to modulate the regenerative and proliferative action of the granular hydrogel composition or the anti-inflammatory action of the granular hydrogel composition, which allows to tune the therapeutic effect of the granular hydrogel composition to a certain type of wound and / or a certain type of patient.

[0023] It is thus a first object of the present invention to provide a granular hydrogel composition for the treatment of wounds, comprising a mixture of a. at least a plurality of first hydrogel microspheres, wherein the first hydrogel microspheres are formed of a first hydrogel scaffold material, and wherein the first hydrogel microspheres further comprise at least a growth factor compound, and b. at least a plurality of second hydrogel microspheres, wherein the second hydrogel microspheres are formed of a second hydrogel scaffold material, and wherein the second hydrogel microspheres further comprise at least a capturing compound for capturing a proinflammatory compound, wherein the capturing compound is bound to the second hydrogel scaffold material, and c. optionally a liquid aqueous phase, characterized in that the plurality of first and second hydrogel microspheres form a pack of hydrogel microspheres and wherein the pack comprises interstitial spaces between the plurality of first and second hydrogel microspheres, which interstitial spaces comprise the liquid aqueous phase, when present.

[0024] It is further a second object of the present invention to provide a granular hydrogel composition according to the first object for use in the treatment of wounds, in particular for use in the treatment of wounds of a diabetic patient or in the treatment of chronic wounds or in the treatment of cutaneous wounds. It is moreover a third object of the present invention to provide a wound dressing, preferably a cutaneous wound dressing, comprising a granular hydrogel composition according to the first object.

[0025] It is moreover a fourth object of the present invention to provide an application device, such as a syringe, at least partially filled with a granular hydrogel composition according to the first object.

[0026] It is lastly a fifth object of the present invention to provide a kit of parts for the preparation of a granular hydrogel composition according to the first object, comprising at least a first container comprising at least a plurality of first hydrogel microspheres, and further optionally a liquid aqueous phase, at least a second container comprising at least a plurality of second hydrogel microspheres, and further optionally a liquid aqueous phase, and optionally a further container comprising a liquid aqueous phase, wherein when neither the first nor the second container comprise a liquid aqueous phase, the liquid aqueous phase is comprised in the further container comprising a liquid aqueous phase, and a device for the mixing of a plurality of first hydrogel microspheres with a plurality of second hydrogel microspheres, and optionally a liquid aqueous phase, such as to obtain the granular hydrogel composition according to the first object.

[0027] Further embodiments of the invention are laid down in the dependent claims.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,

[0030] Fig. 1 shows the concentration of IL-6 in a supernatant when incubated for 5 days in the presence of a granular hydrogel composition according to the present invention, at 5 different weight ratios (100:0, 80:20, 50:50, 20:80, 0:100) of hydrogel microspheres capturing IL-6 (type A, black) and hydrogel microspheres releasing VEGF-A (type H, white). The initial concentration of IL-6 in the supernatant was 1250 ng / mL.

[0031] Fig. 2 shows the concentration of VEGF-A in the supernatant when incubated for 5 days in the presence of a granular hydrogel composition according to the present invention, at 5 different weight ratios (100:0, 80:20, 50:50, 20:80, 0:100) of hydrogel microspheres capturing IL-6 (type A, black) and hydrogel microspheres releasing VEGF-A (type H, white). The initial concentration of IL-6 in the supernatant was 1250 ng / mL.

[0032] Fig. 3 shows the activity of secreted embryonic alkaline phosphatase measured by absorbance (A =630 nm), as a readout of IL-6 signaling activity in transformed HEK IL-6 reporter cells when incubated for 24 h in the presence of conditioned supernatant. The conditioned supernatant was previously incubated with the hydrogel microspheres for 5 days, at 5 different weight ratios (100:0, 80:20, 50:50, 20:80, 0:100) of hydrogel microspheres capturing IL-6 (type A, black) and hydrogel microspheres releasing VEGF-A (type H, white). The initial concentration of IL-6 in the supernatant was 1250 ng / mL.

[0033] Fig. 4 shows the total cell area of GFP-HUVEC cells following incubation for 48 h in the same supernatant as in the sequestration experiment of Fig.1 , in pixel2, as a readout of growth factor activity in the presence of conditioned supernatant. The conditioned supernatant had been previously incubated for 5 days with a granular hydrogel composition according to the present invention, at 5 different weight ratios (100:0, 80:20, 50:50, 20:80, 0:100) of hydrogel microspheres sequestering IL-6 (type A, black) and hydrogel microspheres releasing VEGF- A (type H, white). The initial concentration of IL-6 in the supernatant was 1250 ng / mL.

[0034] DESCRIPTION OF PREFERRED EMBODIMENTS

[0035] It is thus a first object of the present invention to provide a granular hydrogel composition for the treatment of wounds, comprising a mixture of: a. at least a plurality of first hydrogel microspheres, wherein the first hydrogel microspheres are formed of a first hydrogel scaffold material, and wherein the first hydrogel microspheres further comprise at least a growth factor compound, and b. at least a plurality of second hydrogel microspheres, wherein the second hydrogel microspheres are formed of a second hydrogel scaffold material, and wherein the second hydrogel microspheres further comprise at least a capturing compound for capturing a proinflammatory compound, wherein the capturing compound is bound to the second hydrogel scaffold material, and c. optionally a liquid aqueous phase, characterized in that the plurality of first and second hydrogel microspheres form a pack of hydrogel microspheres and wherein the pack comprises interstitial spaces between the plurality of first and second hydrogel microspheres, which interstitial spaces comprise the liquid aqueous phase, when present.

[0036] In the granular hydrogel composition for the treatment of wounds according to the first object of the present invention, the ratio of the plurality of first hydrogel microspheres and the plurality of second hydrogel microspheres can be adjusted in principle to any ratio to best treat a given wound. Thus, in a preferred embodiment of the granular hydrogel composition for the treatment of wounds according to the first object of the present invention the first hydrogel microspheres and second hydrogel microspheres are comprised in the granular hydrogel composition in a predetermined ratio, which may be a number ratio or may be a weight ratio or may be a volume ratio, in particular in a number, weight or volume ratio of between 10:90 and 90:10 and even more preferably between 20:80 and 80:20. In general, the actual ratio will be such that one type of microspheres will be in excess, i.e. the ratio will be more than 50:50 for that microsphere. In that case, the above ranges would range from 50:50 to 90:10 and 50:50 to 10:90, respectively, and preferably range from 50:50 to 80:20 and 50:50 to 20:80, respectively, depending on whether the first hydrogel microspheres or the second hydrogel microspheres are in excess. In one embodiment, the first hydrogel microspheres may be in excess and in another embodiment, the second hydrogel microspheres may be in excess. It should be noted that the term "mixture", in the context of the present invention, relates to a combination of two or more elements in which the two or more elements are intermixed to achieve a mixture, i.e., to achieve an essentially homogenous spatial distribution of both elements within the mixture.

[0037] It will be however understood that the mere ratio between these is not the sole way of controlling the effect of the two populations of microspheres in the wound. Rather, the modulation of both inflammatory signals and proliferative signals may also be influenced by adjusting the in-situ concentration of the active compounds in the microspheres of the population or by choosing ligands having different affinities to the binding partners or by varying the surface- to-volume ratio of microspheres.

[0038] In a preferred embodiment of the granular hydrogel composition for the treatment of wounds according to the first object of the present invention, the granular hydrogel composition is a pharmaceutically acceptable granular hydrogel composition.

[0039] In a preferred embodiment of the granular hydrogel composition for the treatment of wounds according to the first object of the present invention, the granular hydrogel composition for the treatment of wounds, comprises a mixture of which further includes at least a plurality of third hydrogel microspheres, at least a plurality of fourth hydrogel microspheres, and so on. While the main advantage, namely suppression of inflammation and promotion of regeneration of a wounded tissue may be achieved by a mixture that merely incorporates two types of hydrogel microspheres, the invention is not limited to a mixture that merely incorporates two types hydrogel microspheres.

[0040] In a preferred embodiment of the granular hydrogel composition for the treatment of wounds according to the first object of the present invention, the granular hydrogel composition for the treatment of wounds, comprises a mixture of: a. at least a plurality of first hydrogel microspheres, wherein the first hydrogel microspheres are formed of a first hydrogel scaffold material, and wherein the first hydrogel microspheres further comprise at least a growth factor compound, and b. at least a plurality of second hydrogel microspheres, wherein the second hydrogel microspheres are formed of a second hydrogel scaffold material, and wherein the second hydrogel microspheres further comprise at least a capturing compound for capturing a proinflammatory compound, wherein the capturing compound is bound to the second hydrogel scaffold material, and c. optionally a liquid aqueous phase, characterized in that the plurality of first and second hydrogel microspheres form a pack of hydrogel microspheres and wherein the pack comprises interstitial spaces between the plurality of first and second hydrogel microspheres, which interstitial spaces comprise the liquid aqueous phase, when present, and further wherein the mixture further comprises: one or more pluralities of further hydrogel microspheres, with the proviso that the further hydrogel microspheres comprise different growth factors that are different from the growth factor comprised in the first hydrogel microspheres and / or the further hydrogel microspheres comprise different capturing compounds for proinflammatory compounds that are different from the capturing compounds comprised in the second hydrogel microspheres. In such an embodiment, the granular hydrogel composition may be more advantageous in that the selective release of a set of predetermined growth factors and the capture of predetermined proinflammatory compounds results in a more effective wound treatment.

[0041] In a preferred embodiment of the granular hydrogel composition for the treatment of wounds according to the first object of the present invention, the first hydrogel microspheres and second hydrogel microspheres may have about the same or different diameters. The first hydrogel microspheres and / or second hydrogel microspheres may have diameters of between 50 to 500 pm, preferably between 150 and 400 pm.

[0042] In a preferred embodiment of the granular hydrogel composition for the treatment of wounds according to the first object of the present invention the granular hydrogel composition is a flowable composition and may be in the form of a gel or a paste. It will be understood that the actual rheological behavior of the gel will be such that it may be applied, either manually via a tool such as spatula for example or via extrusion from an application device such as a syringe for example. The person skilled in the art will be able to adjust the rheological behavior of the granular hydrogel composition depending on the preferred mode of application, for example by the addition of liquid aqueous phase.

[0043] In a preferred embodiment of the granular hydrogel composition for the treatment of wounds according to the first object of the present invention the treatment of wounds may comprise the treatment of wounds to the skin (cutaneous wounds). While the type of wound is not specially limited, exemplary types of wounds are wounds of the skin, bone trauma, maxillofacial trauma, muscular wounds and so on.

[0044] In a preferred embodiment of the granular hydrogel composition for the treatment of wounds according to the first object of the present invention the treatment of wounds may be wounds that are chronic, or wounds infected by a pathogen. In a preferred embodiment of the granular hydrogel composition for the treatment of wounds according to the first object of the present invention, the granular hydrogel composition may comprise an antibiotic or antiseptic compound, which is preferably releasably bound, either via adsorption or absorption, to a component of the granular hydrogel composition such as for example the first or the second hydrogel microspheres. This may be of advantage when, for example, an infection of the wound is to be treated or prevented.

[0045] In the granular hydrogel composition for the treatment of wounds according to the first object of the present invention, the first hydrogel microspheres are formed of a first hydrogel scaffold material. The hydrogel scaffold material is not particularly limited, and may be chosen from hydrogels such as those based on poly(ethylene glycol), collagen, hyaluronic acid, gelatin methacrylate, alginate, agarose, fibrin, cellulose and other synthetic or natural hydrogel-forming polymers. Suitable hydrogel scaffold material for the first and / or second hydrogel microspheres are, for example, 8-arm poly(ethylene glycol) (PEG) macromers, which may have a Mnof about 10,000 g / mol and are preferably end-functionalized with norbornene (NB).

[0046] It is understood that the first and second hydrogel microspheres are formed of a first or second hydrogel scaffold material, which may be cross-linked or not. In a preferred embodiment of the granular hydrogel composition for the treatment of wounds according to the first object of the present invention, the first and / or second hydrogel scaffold material is / are cross-linked via the addition of a cross-linker. Suitable -linkers are for example, linkers having two or more thiol moieties, such as dithiothreitol (DTT) or PEG di- thiol, which may be used in conjunction with end-functionalized hydrogel scaffold material such as hydrogel scaffold material end-functionalized with norbornene (NB). In this case, the cross-linking reaction may be initiated by the addition of a photoinitiator, such as lithium-phenyl-2,4,6- trimethylbenzoylphosphinate (LAP), and subsequent exposure to radiation such as ultraviolet light. Suitable cross-linkers are for example, linkers having two or more thiol moieties, and can be chosen among linkers having Mnof from about 150 to 2500 g / mol, in particular PEG linkers end-functionalized with thiol.

[0047] In a preferred embodiment, the first and second hydrogel microspheres are formed of a first or second hydrogel scaffold material, wherein the first hydrogel microspheres are formed of a first hydrogel scaffold material that is cross-linked with a first cross-linker and the second hydrogel microspheres are formed of a second hydrogel scaffold material that is crosslinked with a second cross-linker, wherein the first cross-linker has a number average molecular weight (Mn) that is larger than the number average molecular weight (Mn) of the second cross-linker. In a more preferred embodiment, the first cross-linker has a number average molecular weight (Mn) that is larger than the number average molecular weight (Mn) of the second cross-linker, and the first cross-linker has a number average molecular weight (Mn) in the range of 1500 to 5000 g / mol, preferably in the range of 1500 to 2500 g / mol and the second cross-linker has a number average molecular weight (Mn) in the range of 150 to 500 g / mol, preferably in the range of 150 to 250 g / mol. When the first cross-linker has a number average molecular weight (Mn) that is larger than the number average molecular weight (Mn) of the second cross-linker, the mesh size a first hydrogel is increased, and the hydrogel is more permeable for the release of the growth factor and the mesh size of a second hydrogel is decreased and the hydrogel is less permeable for the release of the cytokine.

[0048] In a preferred embodiment, the first and second hydrogel microspheres are formed of a first or second hydrogel scaffold material, wherein the first hydrogel microspheres are formed of a first hydrogel scaffold material that is cross-linked with a first cross-linker and the second hydrogel microspheres are formed of a second hydrogel scaffold material that is crosslinked with a second cross-linker, wherein the first hydrogel scaffold material has a number average molecular weight (Mn) that is larger than the number average molecular weight (Mn) of the second hydrogel scaffold material. When the first hydrogel scaffold material has a number average molecular weight (Mn) that is larger than the number average molecular weight (Mn) of the second hydrogel scaffold material, the mesh size the cross-linked first hydrogel scaffold material is increased and is more permeable for the release of the growth factor and the mesh size the cross-linked second hydrogel is decreased and is less permeable for the release of the cytokine.

[0049] Thus, in general, in the microgranular hydrogel composition of the present invention, the mesh size of the cross-linked first hydrogel scaffold material should preferably be larger than the mesh size of the cross-linked second hydrogel scaffold material. This may for example be achieved by adjusting the number average molecular weight (Mn) of cross-linker and / or the number average molecular weight (Mn) of hydrogel scaffold material in the respective hydrogel microspheres, as provided above.

[0050] The mesh size may alternatively be reflected in the molecular weight between cross-links in a cross-linked hydrogel scaffold material. Thus, in a preferred embodiment, the molecular weight between cross-links in the cross-linked first hydrogel scaffold material is larger than the cross-linked hydrogel scaffold material in the cross-linked second hydrogel scaffold material.

[0051] The first and second hydrogel microspheres are formed of a first or second hydrogel scaffold material and may be provided via any suitable process. As an example, the hydrogel microspheres may be formed of a first hydrogel scaffold material via microfluidic methods such as injection of a pre-gel solution into an oil phase in a microfluidic device or via emulsification methods such as bulk or microfluidic emulsification of a pre-gel solution phase with a droplet generation oil phase.

[0052] In the granular hydrogel composition for the treatment of wounds according to the first object of the present invention, the first hydrogel microspheres further comprise at least a growth factor compound. The growth factor may be adsorbed on the surface of first hydrogel microspheres or may be absorbed in the bulk of the first hydrogel microspheres, when the first hydrogel microspheres are functionalized with a suitable glycosaminoglycan (GAG), to adjust the absorption of the growth factor to the first hydrogel microspheres. For instance, the inclusion of heparin in the first hydrogel microspheres facilitates the absorption of growth factors such as EGF or PDGF on and / or in the first hydrogel microspheres, since growth factors releasably bind to glycosaminoglycans like heparin.

[0053] In the granular hydrogel composition for the treatment of wounds according to the first object of the present invention, the second hydrogel microspheres further comprise at least a capturing compound for capturing a proinflammatory compound. The capturing compound may be adsorbed on the surface of first hydrogel microspheres and / or may be absorbed in the bulk of the second hydrogel microspheres,. It is noted that it is not necessarily required to bind the capturing compound to the second hydrogel microspheres as the binding of the capturing compound to its ligand may be sufficient to inactivate the biological activity of the ligand. Thus, in an alternative embodiment of the granular hydrogel composition for the treatment of wounds according to the first object of the present invention, the capturing compound is releasably bound to the second hydrogel scaffold material, though it is preferred that the capturing compound is non-releasably bound to the second hydrogel scaffold material, for example via a covalent bond such as a thiol ether bond, preferably formed in a thiol-ene reaction, or via a disulfide bond.

[0054] In the granular hydrogel composition for the treatment of wounds according to the first object of the present invention, the capturing compound may in principle, be any suitable compound that can capture the proinflammatory compound via transient interaction or via permanent interaction, i.e. where a covalent chemical bond is formed in a chemical reaction between the capturing compound and the proinflammatory compound. While the chemical nature of the capturing compound is not limited per se, the capturing compound is preferably chosen among polypeptides such as immunoglobulins, polynucleotides such as aptamers, synthetic polymers, or reactive moieties. Conversely, the proinflammatory compound may be any endogenous compound capable of eliciting an inflammatory response and may be chosen among polypeptides and polynucleotides, and preferably is a cytokine such as an interleukin.

[0055] In the granular hydrogel composition for the treatment of wounds according to the first object of the present invention, a liquid aqueous phase may optionally be comprised. The liquid phase may be any suitable liquid and preferably is an aqueous liquid phase such as for example a buffer solution or a physiological saline solution. The liquid phase, when present, fills the interstitial spaces between the microspheres of the granular hydrogel composition and may provide an aqueous phase through which solutes may diffuse with more ease when compared to diffusion across the bulk of the microspheres, which ultimately enhances the exchange of solutes between the granular hydrogel composition and the wound tissue, thereby enhancing the effect of the growth factor compounds that are given off by the first hydrogel microspheres and the enhancing the capturing of inflammatory compounds by the second hydrogel microspheres. Further, the liquid aqueous phase may further comprise biologically active compounds, such as antibiotic, antiseptic, or antimycotic compounds.

[0056] In the granular hydrogel composition for the treatment of wounds according to the first object of the present invention, the first and second hydrogel microspheres form a pack of hydrogel microspheres, i.e. a packed structure of hydrogel microspheres. It is noted that the pack of microspheres provides a higher surface-to-volume ratio to the granular hydrogel composition when compared to a "monolithic" hydrogel composition, thereby further increasing the availability of the bioactive compounds in the granular hydrogel composition and facilitating the diffusion of the bioactive compounds according to the first object of the present invention. To form a pack, after being mixed with each other, which may be achieved via repeatedly aspirating and ejecting a combination of first and second hydrogel microspheres through a pipette or by agitating the combined first and second hydrogel microspheres in vessel. It is noted that in a clinical setting, a double syringe injection system equipped with a static mixer element may also be used to provide a pack of first and second hydrogel microspheres. In a preferred embodiment, the packing fraction may range between 10% to 60%. At packing fractions lower than 10%, the amount of bioactive compounds to be delivered / captured is low, whereas the diffusion of the bioactive compounds is hindered at packing fractions above 60%.

[0057] In a preferred embodiment of the granular hydrogel composition for the treatment of wounds according to the first object of the present invention, the first hydrogel scaffold is a poly(ethylene glycol) hydrogel scaffold and / or where the second hydrogel scaffold is a poly(ethylene glycol) hydrogel scaffold. It has been found that poly(ethylene glycol) hydrogel scaffolds, such as 4 or 8-arm poly(ethylene glycol) (PEG) macromers, allow for good control of particle size of the hydrogel microspheres and / or rheological properties.

[0058] In a preferred embodiment of the granular hydrogel composition for the treatment of wounds according to the first object of the present invention, the first hydrogel scaffold material further incorporates a glycosaminoglycan, and preferably heparin. It is noted that the glycosaminoglycan is preferably chemically modified to allow the non-releasable binding of the glycosaminoglycan to the first hydrogel scaffold, such as for example via the incorporation of thiol groups.

[0059] In a preferred embodiment of the granular hydrogel composition for the treatment of wounds according to the first object of the present invention, the second hydrogel scaffold material incorporates no glycosaminoglycan, and preferably no heparin. While heparin is also able to bind cytokines, the inventors found that it does so indiscriminately, and its inclusion is preferably thus limited to the first hydrogel scaffold material to limit the indiscriminate binding of cytokines of interfering with the specific binding of the cytokines provided via the capturing compounds.

[0060] In a preferred embodiment of the granular hydrogel composition for the treatment of wounds according to the first object of the present invention, the capturing compound comprises at least a proinflammatory compound binding domain for capturing a proinflammatory compound and / or wherein the proinflammatory compound is a cytokine such as an interleukin (IL). It is of note that the binding domain may be any suitable domain that selectively recognizes and binds the proinflammatory compound with high affinity such that it is not immediately released back into the surrounding tissue. In general, the capturing compound should exhibit a Kd in the nanomolar range, preferably of less than 500 nM or in the range of 0.5 to 500 nM and more preferably of less than 100nM or in the range of 0.1 to 100 nM for the proinflammatory compound, cytokine or interleukin (IL). For example, the binding domain may be an antigen-binding site of an immunoglobulin.

[0061] In a preferred embodiment of the granular hydrogel composition for the treatment of wounds according to the first object of the present invention, the capturing compound for capturing a proinflammatory compound is non-releasably bound to the to the second hydrogel scaffold material and preferably is covalently linked to the second hydrogel scaffold material. For example, the capturing compound for capturing a proinflammatory compound is non- releasably bound to the to the second hydrogel scaffold material via click chemistry such as for example a thiol ether bond, preferably formed in a thiol-ene reaction, or via a disulfide bond.

[0062] In a preferred embodiment of the granular hydrogel composition for the treatment of wounds according to the first object of the present invention, the proinflammatory compound binding domain is provided by an immunoglobulin such as an antibody or a scFv, which immunoglobulin is preferably covalently linked to the second hydrogel scaffold material; for example via click chemistry such as for example a thiol ether bond, preferably formed in a thiol-ene reaction, or via a disulfide bond.

[0063] In a preferred embodiment of the granular hydrogel composition for the treatment of wounds according to the first object of the present invention, the proinflammatory compound binding domain is provided via an aptamer, in which the aptamer is designed to specifically bind the proinflammatory compound, and in which the aptamer is preferably covalently linked to the second hydrogel scaffold material. For instance, aptamers may be end-functionalized with thiols, which aptamers may then be used in conjunction with end-functionalized hydrogel scaffold material capable of reacting with the thiol moiety of the aptamer, such as hydrogel scaffold material end-functionalized such as norbornene (NB) and in particular 8-arm poly(ethylene glycol) (PEG) macromers end-functionalized such as norbornene (NB). The inventors have found that in contrast to immunoglobulins, aptamers are more stable in terms of their interaction with their specific ligands over extended time periods, which is one reason why aptamers are particularly preferred.

[0064] In a preferred embodiment of the granular hydrogel composition for the treatment of wounds according to the first object of the present invention, the aptamer will have a Kd in the nanomolar range, preferably of less than 500 nM or in the range of 0.5 to 500 nM and more preferably of less than 100nM or in the range of 0.1 to 100 nM for an interleukin (IL) such as interleukin-6.

[0065] In a preferred embodiment of the granular hydrogel composition for the treatment of wounds according to the first object of the present invention, the growth factor compound is releasably bound to the first hydrogel microspheres via a release compound comprising at least a growth factor compound binding domain for releasably binding the growth factor compound. The affinity of the release compound for the growth factor compound will advantageously have a Kd in the micromolar or millimolar range, preferably of more than 1 pM and more preferably of more than 1 mM.

[0066] In a preferred embodiment of the granular hydrogel composition for the treatment of wounds according to the first object of the present invention, the growth factor compound is VEGF or PDGF.

[0067] In a preferred embodiment of the hydrogel composition for the treatment of wounds according to the first object of the present invention, the release compound comprising at least a growth factor compound binding domain is preferably non-releasably, or covalently, bound to the first hydrogel scaffold material. The chemical nature of the release compound is not particularly limited and may be chosen among antibodies or aptamers, for example. In particular, the release compound is an antibody or an aptamer, which releasably bind the at least one growth factor such as VEGF or PDGF. For instance, an aptamer may be end- functionalized with thiols, which aptamer may then be used in conjunction with end- functionalized hydrogel scaffold material capable of reacting with the thiol moiety of the aptamer, such as hydrogel scaffold material end-functionalized with norbornene (NB) and in particular 8-arm poly(ethylene glycol) (PEG) macromers end-functionalized with norbornene (NB) to provide a thiol ether bond between the aptamer and the first hydrogel scaffold material.

[0068] It is further a second object of the present invention to provide a granular hydrogel composition according to the first object for use in the treatment of wounds, in particular for use in the treatment of wounds of a diabetic patient or in the treatment of chronic wounds or in the treatment of cutaneous wounds.

[0069] It is moreover a third object of the present invention to provide a wound dressing, preferably a cutaneous wound dressing, comprising a granular hydrogel composition according to the first object. It is moreover a fourth object of the present invention to provide an application device, such as a syringe, at least partially filled with a granular hydrogel composition according to the first object.

[0070] It is lastly a fifth object of the present invention to provide a kit of parts for the preparation of a granular hydrogel composition according to the first object, comprising at least a first container comprising at least a plurality of first hydrogel microspheres, and further optionally a liquid aqueous phase, at least a second container comprising at least a plurality of second hydrogel microspheres, and further optionally a liquid aqueous phase, and optionally a further container comprising a liquid aqueous phase, wherein when neither the first nor the second container comprise a liquid aqueous phase, the liquid aqueous phase is comprised in the further container comprising a liquid aqueous phase, and a device for the mixing of a plurality of first hydrogel microspheres with a plurality of second hydrogel microspheres, and optionally a liquid aqueous phase, such as to obtain the granular hydrogel composition according to the first object.

[0071] EXPERIMENTAL DATA

[0072] Different granular hydrogel compositions comprising a mixture of a first population of first hydrogel microspheres (type H, o) incorporating 8-arm PEG-norbornene (PEG-NB) scaffolds crosslinked with SH-PEG-SH and having SH- heparin coupled to them and loaded with VEGF-A and a second population of second hydrogel microspheres (type A, •) incorporating 8- arm PEG-norbornene (PEG-NB) scaffolds crosslinked with DTT and having SH-aptamers against IL-6 coupled to them, were produced at different weight ratios between type A and H, namely at 100:0, 80:20; 50:50, 20:80 and 0:100.

[0073] The type H heparin functionalized microgels were functionalized with SH-heparin (2.5 pM) and loaded with 500 ng / mL VEGF-A solution overnight. The type A aptamer functionalized microgels were functionalized with SH-aptamer (2.5 pM).

[0074] A release solution for VEGF-A consisting of 1250 ng / mL IL-6 was added on the different granular hydrogel compositions and kept for 5 days at 37 °C with mechanical agitation and the concentration of IL-6 and VEGF-A was measured in the supernatant (Fig. 1 and 2). Further, in a separate experiment, the same supernatant was applied to two types of cells (HEK cells / GFP-HUVECS) to determine the effect on said cells of the capture of IL-6 from the supernatant and the release of VEGF-A into the supernatant (Fig. 3 and 4).

[0075] In the HEK cells, as shown in Fig. 3, the secreted embryonic alkaline phosphatase (SEAP) activity measured by absorbance (A = 630 nm) as a readout of IL-6 signaling activity in transformed HEK IL-6 reporter cells (P < 0.001 for 1250 ng / mL IL-6), as shown in Fig. 3, shows that for higher fractions of type A aptamer functionalized microgels, the IL-6 signalling activity as evidenced by SEAP absorbance is lowered. This is attributable to lower levels of available IL-6 in the supernatant.

[0076] In the GFP-HUVEC cells which are dependent on VEGF-A for survival when plated on 2D substrates in the absence of any supporting matrix or supporting stromal cells, as shown in Fig. 4, the total cell area of GFP-HUVEC cells following incubation with the supernatant from the sequestration / release experiment for 5 days (P< 0.0001 for 1250 ng / mL IL-6) increased (indicating growth) when the fraction of type B increased.

[0077] Thus, the above results demonstrate that the granular hydrogel compositions can simultaneously on one hand reduce and modulate inflammatory response dependent on the amount of the respective hydrogel microspheres by capturing interleukins from the surrounding environment and on the other hand, promote and modulate proliferative response dependent on the amount of the respective hydrogel microspheres by releasing growth factors into the surrounding environment.

[0078] Synthesis of 8-arm PEG-NB:

[0079] The synthesis of 8-arm PEG-norbornene (PEG-NB) was done as published previously (D. B. Emiroglu, A. Bekcic, D. Dranseikiene, X. Zhang, T. Zambelli, A. J. deMello, M. W. Tibbitt, Sci. Adv. 2022, 8). Briefly, 8-arm PEG-NH2HCI (Mn = 10 000 g mol-1 ; 8 g, 0.8 mmol PEG, 6.4 mmol NH2) was dissolved in anhydrous dimethylformamide (DMF; 5 mL). First, N,N- Diisopropylethylamine (DIPEA; 4.46 mL, 25.6 mmol, 4 eq.) and 1- [bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate] (HATLI; 4.86 g, 12.8 mmol, 2 eq.) were added to the PEG solution. Next, 5-norbornene-2-carboxylic acid; (3.12 ml, 25.6 mmol, 4 eq.; Sigma) was added to the mixture and stirred overnight at room temperature (RT). The reaction mixture was precipitated twice in diethyl ether at 4°C and the precipitate was dialyzed in a regenerated cellulose dialysis membrane against dbhO for 3 days. Finally, the polymer solution was frozen and lyophilized.

[0080] Fabrication of microgels via rotisserie emulsification:

[0081] Pre-gel solutions with DTT (Mw = 154 g mol-1) or PEG di-thiol (PEG-dSH) (Mn = 2000 g mol-1) were prepared for the aptamer-functionalized and heparin-functionalized microgels, respectively. In an Eppendorf tube, pre-gel solutions and five-fold excess droplet generation oil (BioRad) were added. The Eppendorf tube oil was placed on a Thermo Scientific tube revolver (Thermo Scientific). The solution was rotated at 30 ref for 90 s before being exposed to UV light for 90 s (A = 365 nm, 1 = 15 mW cm-2). Cross-linked microgels were centrifuged at 16000 ref for 5 min, and then the supernatant was aspirated. The gels were washed twice with 0.0125 wt% Tween20 in PBS at a 5-fold excess, and twice with PBS (5- fold excess) by centrifugation between each wash as described above.

[0082] Aptamer folding and disulfide reduction:

[0083] Reconstituted IL-6-aptamer solutions (100 pM) were diluted in folding buffer (Cambio UK) at 10x working concentration. The solution was then heated to 95 °C for 5 min to ensure proper folding and allowed to cool to room temperature. Folded aptamers were diluted 5- fold to working concentration in equal volume of 10 mM TCEP and incubated for 10 min at room temperature to reduce disulfide bonds and expose thiol groups or hydrogel attachment. Final working solutions at 5 pM were prepared by diluting reduced aptamers to a solution containing and 5 mM MgCh in PBS.

[0084] Aptamer attachment to hydrogels:

[0085] Equal volume of aptamer working solution was added to the microgels to achieve a final concentration of 2.5 pM in the gels. The mixture was placed on a shaker plate and allowed to incubate overnight. 10 mM TCEP and 0.2 wt% LAP was then added to the mixture and exposed to UV light for 90 sec (A = 365 nm, 1 = 15 mW / cm2). Functionalized microgels were washed using 5-fold excess MgCh (1 mM in PBS), with centrifugation at 16000 ref for 5 min, for a total of four washes.

[0086] VEGF-A loading and release assays:

[0087] PEG-dSH-containing granular hydrogels were prepared using the rotisserie emulsification method as mentioned above. Equal volume of 5 pM heparin-thiol (heparin-SH, MW 27 kDa) solution was added to the gels. The gels were then protected from direct exposure to light and incubated overnight under mechanical agitation at room temperature. The next day, 10 mM TCEP was added to reduce any dimerized heparin, and the gels were incubated for 30 min. 0.2 wt% LAP was then added, and the heparin-SH was attached to the gels via exposure to UV light for 5 min (A = 365 nm, 1 = 15 mWcm-2). The gels were then centrifuged at 16000 ref for 5 min and the supernatant was removed. 5-fold excess volume of PBS was added to wash the gels for a total of four washes, centrifuging as mentioned above between each wash. VEGF-A at 500 ng mL-1was prepared in PBS and added to the gels, with 4-fold excess volume ratio. The gels were allowed to sequester the VEGF-A overnight at room temperature under agitation.

[0088] Simultaneous release and sequestration assays:

[0089] Granular hydrogels were prepared as mentioned above. DTT-containing microgels were functionalized with IL-6 aptamer for IL-6 sequestration (termed A microgels) and PEG-dSH- containing microgels were heparin-SH functionalized and loaded with 500 ng mL-1 VEGF- A (termed H microgels). Once prepared, the two microgel populations were mixed together in an Eppendorf tube using a positive displacement pipette at five different weight ratios: 100% A / 0% H, 80% A / 20% H, 50% A / 50% H, 20% A / 80% H, 0% A / 100%. The microgels were then transferred to a 48 well-plate, and 4-fold excess volume of IL-6 containing media (1250 ng mL-1 IL-6 in PBS + 1% BSA +1% penicillin / streptomycin) was added to initiate IL- 6 sequestration and VEGF-A release. The plate was incubated at 37 °C in a humidified atmosphere at 5% CO2 under agitation at 90 rpm. Timepoints were collected at 120 h, and samples were either stored at -80°C until quantification or used immediately for cell-based assays.

[0090] HEK-Blue cell assays: Human embryonic kidney (HEK) IL-6 sensor cells were purchased from Invivogen (Cat # hkb-hil6). These cells are transfected with a reporter gene expressing secreted alkaline phosphatase (SEAP) under the control of a STAT3 sensitive promoter. With IL-6 signaling, the JAK-STAT pathway is activated leading to SEAP expression. SEAP can be detected by absorbance assays. HEK-Blue cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (4.5 g L-1Glucose) (Gibco) supplemented with 10% heat- inactivated fetal bovine serum (FBS) (Gibco), and 1% PenStrep. During the growth phase, normocin and selection antibiotics were added to the media (removed for the testing). For testing IL-6 activity, HEK- Blue cells were seeded at 50’000 cells per well in a 96-well plate. Supernatant from the sequestration and release assay was diluted in media to be in the detectable range and added to the wells (1 :10 v / v). Cells were incubated with this supernatant for 24 h. 20 pl supernatant was collected and added to 180 pl of QuantiBlue detection reagent (Invivogen, rep-qbs). The mixture was incubated for 1-3 h at 37 °C after which the absorbance was measured at A = 637 nm.

[0091] HUVEC growth assays:

[0092] Green fluorescent protein labelled human umbilical vascular endothelial cells (GFP- HUVECs) were cultured in Modified Eagle Medium a (Sigma), supplemented with 10% FBS, Glutamax (Gibco) and 50 ng mL-1fibroblast growth factor 2 (FGF-2) on gelatin-coated flasks. Cells were seeded at 10’000 cells per well on a non-coated 96-well plate in media (without FGF-2) with supernatants from the sequestration and release experiments. The supernatant was supplemented with 10% FBS before applying it to the cells. Cell growth and spreading was measured at 48 h by fluorescent imaging of GFP signal in the whole well. Total cell spread area in px2was measured with Imaged.

Claims

CLAIMS1. A granular hydrogel composition, preferably for the treatment of wounds, comprising a mixture of a. at least a plurality of first hydrogel microspheres, wherein the first hydrogel microspheres are formed of a first hydrogel scaffold material, and wherein the first hydrogel microspheres further comprise at least one growth factor compound, and b. at least a plurality of second hydrogel microspheres, wherein the second hydrogel microspheres are formed of a second hydrogel scaffold material, and wherein the second hydrogel microspheres further comprise at least a capturing compound for capturing a proinflammatory compound, wherein the capturing compound is bound to the second hydrogel scaffold material, and c. optionally a liquid aqueous phase, characterized in that the plurality of first and second hydrogel microspheres form a pack of hydrogel microspheres and wherein the pack comprises interstitial spaces between the plurality of first and second hydrogel microspheres, which interstitial spaces comprise the liquid aqueous phase, when present, wherein the proinflammatory compound binding domain is provided via an aptamer, which aptamer is covalently linked to the second hydrogel scaffold material.

2. The granular hydrogel composition for the treatment of wounds according to claim 1 , wherein the first hydrogel scaffold and the second hydrogel scaffold are crosslinked and the mesh size of the cross-linked first hydrogel scaffold material is larger than the mesh size of the cross-linked second hydrogel scaffold material.

3. The granular hydrogel composition for the treatment of wounds according to claim 1 or 2, wherein the first hydrogel scaffold is a poly(ethylene glycol) hydrogel scaffold and / or where the second hydrogel scaffold is a poly(ethylene glycol) hydrogel scaffold.

4. The granular hydrogel composition for the treatment of wounds according to any one claim 1 to 3, wherein the first hydrogel scaffold material further incorporates a glycosaminoglycan, and preferably heparin, as a release compound for the at least one growth factor, and / or wherein the second hydrogel scaffold material incorporates no glycosaminoglycan, and preferably no heparin.

5. The granular hydrogel composition for the treatment of wounds to any one claim 1 to 4, wherein the capturing compound comprises at least a proinflammatory compound binding domain for capturing a proinflammatory compound and / or wherein the proinflammatory compound is a cytokine such as an interleukin (IL).

6. The granular hydrogel composition for the treatment of wounds according to any one claim 1 to 5, wherein growth factor compound is releasably bound to the first hydrogel microspheres via a release compound comprising at least a growth factor compound binding domain for releasably binding the growth factor compound, which release compound is preferably chosen among antibodies or aptamers.

7. The granular hydrogel composition for the treatment of wounds according to any one claim 1 to 6, wherein growth factor compound is FGF, TGF-p ,VEGF or PDGF.

8. The granular hydrogel composition for the treatment of wounds according to any one claim 1 to 7, wherein the release compound comprising at least a growth factor compound binding domain is preferably covalently linked to the first hydrogel scaffold material, preferably via a thiol ether bond.

9. The granular hydrogel composition according to any one claim 1 to 8, for use in the treatment of wounds, in particular for use in the treatment of wounds of a diabetic patient or in the treatment of chronic wounds, and in particular cutaneous wounds.

10. The granular hydrogel composition for use in the treatment of wounds according to claim 9, wherein the granular hydrogel composition is comprised in a wound dressing.

11. A wound dressing, preferably a cutaneous wound dressing, comprising a granular hydrogel composition according to any one claim 1 to 8.

12. An application device, such as a syringe, at least partially filled with a granular hydrogel composition according to any one claim 1 to 8.

13. A kit of parts for the preparation of a granular hydrogel composition according to any one claim 1 to 8, comprising at least a first container comprising a volume of at least a plurality of first hydrogel microspheres, and further optionally a liquid aqueous phase, at least a second container comprising a volume of at least a plurality of second hydrogel microspheres, and further optionally a liquid aqueous phase, and optionally a further container comprising a volume of a liquid aqueous phase, wherein when neither the first nor the second container comprise a liquid aqueous phase, the liquid aqueous phase is comprised in the further container comprising a liquid aqueous phase, a device for the mixing of a plurality of first hydrogel microspheres with a plurality of second hydrogel microspheres, optionally in a predefined ratio, and optionally a liquid aqueous phase, such as to obtain the granular hydrogel composition according to any one claim 1 to 8.

Images