CROSSLINKABLE HYDROGEL COMPOSITIONS

Abstract

A cross-linkable hydrogel composition comprising an aqueous base carrier composition and a cross-linkable hydrogel precursor, wherein the aqueous base carrier composition comprises one or more polymers and one or more nanoparticles, wherein the one or more polymers are selectively adsorbed to the one or more nanoparticles, and wherein the one or more polymers and the one or more nanoparticles form a shear-thinning and self-healing hydrogel and wherein the one or more polymers and the one or more nanoparticles and cross-linkable hydrogel precursor are each comprised in the cross-linkable hydrogel composition in a concentration at which neither the one or more polymers nor the nanoparticles, taken alone, form a hydrogel, characterized in that the cross-linkable hydrogel precursor is not selectively adsorbed to the one or more nanoparticles and in that the cross-linkable hydrogel precursor and the one or more nanoparticles don't form a transient shear-thinning and self-healing hydrogel.

Claims

1. A cross-linkable hydrogel composition comprising an aqueous base carrier composition and a cross-linkable hydrogel precursor, wherein the aqueous base carrier composition comprises one or more polymers and one or more nanoparticles, wherein the one or more polymers are selectively adsorbed to the one or more nanoparticles, and wherein the one or more polymers and the one or more nanoparticles form a shear-thinning and self-healing hydrogel and wherein the one or more polymers and the one or more nanoparticles are each comprised in the cross-linkable hydrogel composition in a concentration at which neither the one or more polymers nor the nanoparticles, taken alone, form a hydrogel, characterized in that the cross-linkable hydrogel precursor is not selectively adsorbed to the one or more nanoparticles and in that the cross-linkable hydrogel precursor and the one or more nanoparticles do not form a shear-thinning and self-healing hydrogel and further in that the cross-linkable hydrogel precursor is comprised in the cross-linkable hydrogel composition in a concentration at which the cross-linkable hydrogel precursor, taken alone, does not form a hydrogel.

2. The cross-linkable hydrogel composition according to claim 1, wherein the one or more nanoparticles have particle size D.sub.h of between 20 nm and 100 nm, or the nanoparticles are present in an amount of between 1 and 25 wt % based on the weight of the cross-linkable hydrogel composition, or wherein the one or more nanoparticles have particle size D.sub.h of between 20 nm and 100 nm and the nanoparticles are present in an amount of between 1 and 25 wt % based on the weight of the cross-linkable hydrogel composition.

3. The cross-linkable hydrogel composition according to claim 1, wherein the one or more polymer of the aqueous base carrier composition is one or more semisynthetic polysaccharide.

4. The cross-linkable hydrogel composition according to claim 1, wherein the one or more polymer of the aqueous base carrier composition has a molecular weight of from 100 kDa to 850 kDa.

5. The cross-linkable hydrogel composition according to claim 1, wherein it further comprises one or more active pharmaceutical ingredients.

6. The cross-linkable hydrogel composition according to claim 1, wherein it further comprises one or more cells.

7. The cross-linkable hydrogel composition according to claim 1, wherein the cross-linkable hydrogel precursor is cross-linkable by radiation, change of pH, change of temperature or by contact with a crosslinking agent.

8. The cross-linkable hydrogel composition according to claim 1, wherein the cross-linkable hydrogel composition further comprises a photoinitiator capable of cross-linking the cross-linkable hydrogel precursor upon exposure to a cross-linking radiation.

9. The cross-linkable hydrogel composition according to claim 1, wherein the cross-linkable hydrogel precursor is chosen from animal or plant homo- or heteropolysaccharides.

10. The cross-linkable hydrogel composition according to claim 1, wherein the cross-linkable hydrogel precursor is a protein.

11. The cross-linkable hydrogel composition according to claim 1, wherein the cross-linkable hydrogel precursor is a synthetic polymer.

12. (canceled)

13. (canceled)

14. An object comprising a cross-linked cross-linkable hydrogel composition according to claim 1.

15. A process of manufacturing an object comprising the steps of a. providing a cross-linkable hydrogel composition according to claim 1, b. obtaining a green body comprising the cross-linkable hydrogel composition c. cross-linking or photo-cross-linking the green body to form the object or a part thereof.

16. The cross-linkable hydrogel composition according to claim 1, wherein the one or more nanoparticles have particle size D.sub.h of between 45 nm and 65, or the one or more nanoparticles are present in an amount of between 10 and 20 wt % based on the weight of the cross-linkable hydrogel composition, or wherein the one or more nanoparticles have particle size D.sub.h of between 45 nm and 65 and the one or more nanoparticles are present in an amount of between 10 and 20 wt % based on the weight of the cross-linkable hydrogel composition.

17. The cross-linkable hydrogel composition according to claim 1, wherein the one or more polymer of the aqueous base carrier composition is selected from the group consisting of semisynthetic cellulose ethers or mixtures thereof.

18. The cross-linkable hydrogel composition according to claim 1, wherein the one or more polymer of the aqueous base carrier composition is selected from the group consisting of methyl cellulose, ethyl cellulose, ethyl methyl cellulose, hydroxypropylmethyl cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, or mixtures thereof.

19. The cross-linkable hydrogel composition according to claim 1, wherein the one or more polymer of the aqueous base carrier composition is hydroxypropylmethyl cellulose, hydroxyethyl cellulose, or mixtures thereof.

20. The cross-linkable hydrogel composition according to claim 1, wherein it further comprises one or more microorganism cells.

21. The cross-linkable hydrogel composition according to claim 1, wherein it further comprises one or more active pharmaceutical ingredients, wherein the one or more active pharmaceutical ingredients are confined to the one or more nanoparticles of the base carrier composition.

22. The cross-linkable hydrogel composition according to claim 1, wherein it further comprises one or more microorganism cells.

23. The cross-linkable hydrogel composition according to claim 1, wherein the cross-linkable hydrogel precursor is chosen from alginate and derivatives thereof, or from methacrylated or acrylated derivatives of hyaluronic acid.

24. The cross-linkable hydrogel composition according to claim 1, wherein the cross-linkable hydrogel precursor is collagen.

25. The cross-linkable hydrogel composition according to claim 1, wherein the cross-linkable hydrogel precursor is a poly(ethylene glycol) diacrylate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0051] 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,

[0052] FIG. 1 shows grid structures (8×8 mm) including a colorant bound to nanoparticles that were DIW printed using solely the aqueous base carrier composition, top row, and using the cross-linked cross-linkable hydrogel composition comprising both the aqueous base carrier composition and cross-linkable hydrogel precursor in the form of PEGDA (Example 4), bottom row.

[0053] FIG. 2 shows the mass fraction of released colorant over a time of 5 days into an aqueous buffer from the grid structures of cross-linked cross-linkable hydrogel composition (Example 4), dotted line, and from the sole aqueous base carrier composition, dashed line.

[0054] FIG. 3 shows a) G′ as well as G″ over a frequency range (ω=100-0.1 rad s.sup.−1, γ=0.3%, 25° C.) for compositions according to Examples 1 to 4 and for the sole aqueous base carrier composition, which was measured at 4° C.; b) Shear rate ramp (δγ/δt=0.1-100 s.sup.−1) analysis for compositions according to Examples 1 to 4 and for the sole aqueous base carrier composition; c) Step strain analysis with alternating intervals at low (γ=0.3%) and high (γ=1000%) shear strain amplitude at constant angular frequency (ω=10 rad s-1) for compositions according to Examples 1 to 4 and for the sole aqueous base carrier composition; d) self-healing was confirmed by plotting the loss factor (tan δ=MG′) during the step strain analysis.

[0055] FIG. 4 shows a) grid structures (8×8 mm) that were DIW printed with the cross-linkable hydrogel compositions according to Examples 1-4 and solely base aqueous carrier composition, center. a straight needle was used at a constant velocity. 4b) indentation tests made on cross-linked cross-linkable hydrogel compositions according to Examples 1-4 and sole base carrier composition (UNI-15) after equilibration in PBS (pH 7.4, 37° C.). The indentation test could not be performed on sole base carrier composition (UNI-15).

EXAMPLES

[0056] Materials

[0057] Hydroxypropylmethylcellulose (HPMC; M.sub.n˜700 kDa, Ref. H3785), methacrylic anhydride (MA; Ref 276685), 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (Hepes; 99%, Ref H3375), Oil Red O (OR; Ref 00625), calcium chloride (CaCl2; Ref 21074), anti-mouse IgG-Atto 594 (IgG-Atto 594; Ref 76085), 2,4,6-trimethylbenzoyl chloride (Ref 682519), dimethyl phenylphosphonite (Ref 149470), lithium bromide (Ref 213225), DAPI (Ref D9542) and 2-butanone (Ref 360473) were purchased from Sigma-Aldrich (Buchs, Switzerland). PEG5k-b-PLA16k (AK054) and PEG5k-b-PLA14k (AK049) were purchased from Akina (West Lafayette, Ind., USA). Sodium alginate (Alg; Grade I-1G-80) was purchased from Kimica International (Tokyo, Japan); poly(ethylene glycol) diacrylate (PEGDA; Mw ˜10 kDa, Ref ACRL-PEG-ACRL-10K) was purchased from Laysan Bio Inc. (Arab, Ala., USA). Sodium hyaluronate (HA; Mw ˜700 kDa, Ref HA700K) was purchased from Lifecore (Chaska, Minn., USA). Collagen (Col; 10 mg/ml, Ref 5133-20ML) was purchased from Advanced BioMatrix (San Diego, Calif., USA). Acetone (Ref 20066.296) and acetonitrile (Ref 20071.294) were purchased from VWR Chemicals. Low glucose DMEM (Ref 22320-022), Pen Strep (PS, Ref 15140-122), FGF (Ref PHG0026), and fetal bovine serum (FBS; Ref 10270106), LIVE/DEAD Viability/Cytotoxicity kit (Ref L3224), AlexaFluor 488 Phalloidin (Ref A12379) were purchased from ThermoFisher.

[0058] Preparation of PEG-b-PLA Nanoparticles

[0059] The PEG-b-PLA nanoparticles were obtained by dissolving 140 mg PEG-b-PLA in 2 ml of acetone, from which solution the PEG-b-PLA nanoparticles were nanoprecipitated by dropwise addition (15 μL every 3 s) of said solution into 10 ml Milli-Q water under a high stir rate (650 rpm). After overnight evaporation of acetone, the thus obtained PEG-b-PLA nanoparticles were filtered by ultracentrifugation and re-suspended to a final concentration of 20 wt % in aqueous buffer (20 mM HEPES and 275 mM mannitol) or [for UNI, UNI-PEGDA, UNI-MeHA, and UNI-Alg] or to a final concentration of 25 wt % [for UNI-Col)] in Milli-Q water.

[0060] The size of the PEG-b-PLA nanoparticles (D.sub.h˜55 nm) and dispersity of the PEG-b-PLA nanoparticles (PDI ˜0.1) were characterized by dynamic light scattering on a Malvern ZetaSizer Nano ZS at a scattering angle of 173° and at 25° C.).

[0061] Preparation of Base Carrier Composition Using PEG-b-PLA Nanoparticles and HPMC (UNI-15)

[0062] An aqueous base carrier composition of HPMC and PEG-b-PLA nanoparticles was prepared by first dissolving HPMC in buffer solution (20 mM HEPES and 150 mM D-mannitol, pH=7.4) at 1 wt % and allowed to equilibrate overnight (25° C.). Thereafter, the HPMC solution was combined with the 20 wt % nanoparticles in aqueous buffer (20 mM HEPES and 275 mM mannitol) to yield a base carrier composition of 15 wt % PEG-b-PLA nanoparticles and 1 wt % HPMC.

Example 1: Preparation of a Cross-Linkable Hydrogel Composition with PEG-b-PLA Nanoparticles and HPMC as Hydrogel-Forming Polymer with Alginate as Cross-Linkable Hydrogel Precursor (UNI-Alg)

[0063] A composition was prepared by first dissolving 1 wt % HPMC in an aqueous buffer (20 mM HEPES and 150 mM D-mannitol, pH=7.4) together with 1 wt % alginate and allowed to equilibrate overnight (25° C.). This composition comprising 1 wt % alginate and HPMC, respectively, was then combined and mixed with the above suspension of 20 wt % PEG-b-PLA nanoparticles in aqueous buffer (20 mM HEPES and 275 mM mannitol) such as to provide for the cross-linkable hydrogel composition according to the present invention comprising 1 wt % alginate, 15 wt % PEG-b-PLA nanoparticles and 1 wt % HPMC.

Example 2: Preparation of of a Cross-Linkable Hydrogel Composition with PEG-b-PLA Nanoparticles and HPMC as Polymer with Methacrylate Hyaluronic Acid as Cross-Linkable Hydrogel Precursor (UNI-MeHA)

[0064] An aqueous base carrier composition according to the present invention comprising 1 wt % methacrylate hyaluronic acid, 15 wt % PEG-b-PLA nanoparticles and 1 wt % HPMC was obtained in the same manner as above, with the exception that 1 wt % methacrylate hyaluronic acid was dissolved together with 1 wt % HPMC and 0.1 wt % lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as photoinitiator in an aqueous buffer (20 mM HEPES and 150 mM D-mannitol, pH=7.4).

[0065] The Methacrylate Hyaluronic had a Degree of Substitution of about 0.4.

Example 3: Preparation of of a Cross-Linkable Hydrogel Composition with PEG-b-PLA Nanoparticles and HPMC as Polymer with Poly(Ethylene Glycol) Diacrylate (PEGDA) as Cross-Linkable Hydrogel Precursor(UNI-PEGDA)

[0066] An aqueous base carrier composition according to the present invention comprising 1 wt % poly(ethylene glycol) diacrylate, 15 wt % PEG-b-PLA nanoparticles and 5 wt % HPMC was obtained in the same manner as above, with the exception that 1 wt % poly(ethylene glycol) diacrylate was dissolved together with 5 wt % HPMC and 0.1 wt % lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as photoinitiator in an aqueous buffer (20 mM HEPES and 150 mM D-mannitol, pH=7.4).

Example 4: Preparation of a Cross-Linkable Hydrogel Composition with PEG-b-PLA Nanoparticles and HPMC as Polymer with Collagen as Cross-Linkable Hydrogel Precursor (UNI-Col)

[0067] A composition was prepared by first dissolving 1 wt % HPMC in Milli-Q water and allowed to equilibrate overnight (4° C.). This composition comprising 1 wt % HPMC was then combined and mixed with the above suspension of 25 wt % PEG-b-PLA nanoparticles in Milli-Q water such as to provide for the aqueous base carrier composition of the cross-linkable hydrogel composition according to the present invention comprising 1 wt % alginate, 15 wt % PEG-b-PLA nanoparticles and 1 wt % HPMC.

[0068] Collagen was combined and mixed with the aqueous base carrier composition and until a concentration of 0.2 wt % collagen, 1 wt % HPMC and 15 wt % PEG-b-PLA nanoparticles reached.

Example 5: Preparation of of a Cross-Linkable Hydrogel Composition with Silica Nanoparticles and HEC as Polymer with Methacrylate Dextran as Cross-Linkable Hydrogel Precursor

[0069] A composition was prepared by first dissolving 1 wt % HEC in Milli-Q water and allowed to equilibrate overnight (4° C.). This composition comprising 1 wt % HEC was then combined and mixed a suspension of 25 wt % silica nanoparticles in Milli-Q water such as to provide for the aqueous base carrier composition of the cross-linkable hydrogel composition according to the present invention comprising 3 wt % methacrylate dextran, 15 wt % silica nanoparticles and 1 wt % HEC.

[0070] The methacrylate dextran had a degree of substitution of about 0.7.

[0071] While the composition exhibited desirable shear thinning and self-healing, the shelf life was limited in the sense that the composition would phase-separated within 24 hrs whereas with the other exemplary compositions no phase separation could be observed even after weeks or months.

[0072] Cross-Linking of the Cross-Linkable Hydrogel Compositions

[0073] The alginate cross-linkable hydrogel composition of Example 1 was cross-linked by immersing the scaffold in 100 mM CaCl.sub.2 solution for 30 min.

[0074] The PEGDA and MeHA cross-linkable hydrogel compositions of Example 2 and 3 were cross-linked with visible light (k=405 nm, at I=15 mW cm.sup.−2) for 5 min.

[0075] The collagen cross-linkable hydrogel composition of Example 4 was cross-linked by placing in an incubator (37° C., 5% CO.sub.2) for 120 min.

[0076] Results

[0077] As can be seen from FIG. 1, grid structures (8×8 mm) including a colorant bound to nanoparticles were DIW printed using solely aqueous base carrier composition as described above and using the cross-linked cross-linkable hydrogel composition comprising both the aqueous base carrier composition and cross-linkable hydrogel precursor in the form of PEGDA (Example 4). A rapid erosion was observed for the grid structures of sole aqueous base carrier composition, whereas for the grid structures of cross-linked cross-linkable hydrogel composition maintained its structure and released colorant over a longer time in the aqueous buffer.

[0078] As can be seen from FIG. 2, the above grid structures of aqueous base carrier composition released the entire colorant over 5 days into an aqueous buffer whereas the grid structures of cross-linked cross-linkable hydrogel composition (Example 4) exhibited an initial burst release of ˜35% on day 1, followed by minimal release as the grid structures slowly eroded.

[0079] Thus, the cross-linkable hydrogel compositions according to the present invention exhibit extended stability and structural integrity in aqueous environments.

[0080] As can be seen from FIG. 3 (3a/b/c/d), the cross-linkable hydrogel compositions according to the present invention of Examples 1-4 a) formed viscoelastic hydrogels, where G′>G″ over the whole frequency range (ω=100-0.1 rad s.sup.−1, γ=0.3%, 25° C.; the same behavior was observed for the aqueous base carrier composition, which was measured at 4° C.; b) Shear rate ramp (δγ/δt=0.1-100 s-1) analysis showed that shear-thinning behavior was maintained with the inclusion of each cross-linkable hydrogel precursor; c) Step strain analysis with alternating intervals at low (γ=0.3%) and high (γ=1000%) shear strain amplitude at constant angular frequency (ω=10 rad s.sup.1) showed rapid and repeatable self-healing was maintained (˜80% recovery in less than 60 sec) of each cross-linkable hydrogel precursor; d) self-healing was confirmed by plotting the loss factor (tan δ=G″/G′) during the step strain analysis.

[0081] As can be seen from FIG. 4a) grids (8×8 mm) were DIW printed with the cross-linkable hydrogel compositions according to the present invention of Examples 1-4 and solely base carrier composition. In all cases, a straight needle was used at a constant velocity. The pressure applied varied between ˜60 kPa for the sole base carrier composition and the cross-linkable hydrogel composition of Example 4, to −80 kPa for the cross-linkable hydrogel composition of Example 1, 2 and 3. The high printing fidelity illustrates the robustness of the cross-linkable hydrogel compositions; 4b) indentation tests on cross-linked cross-linkable hydrogel compositions of Examples 1-4 and sole base carrier composition (UNI) after equilibration in PBS (pH 7.4, 37° C.). The indentation test could not be performed on sole base carrier composition (UNI).