Biomimetic networks comprising polyisocyanopeptide hydrogels

Abstract

A polymer hydrogel having a polymer formed by the crosslinking reaction of a polymeric unit A according to formula (I), ##STR00001##
with a crosslinking unit B according to formula (II) ##STR00002##
and water, wherein n=100-10,000, preferable 250-2500, more preferable 500-1500; m=independently 2-10, preferably 3 or 4; FG is a functional moiety that can be covalently coupled to the complementary functional moiety F1 or F2 of the crosslinking unit (B); k=0.01-0.05; h=0, 1 or 2; the spacer is an organic moiety, having a main chain comprising at least two functional moieties F1 and F2, wherein the length of the crosslinker in the extended conformation as determined by molecular modeling (including spacer and functional groups F1 and F2) is between 2.5 and 12 nm, or wherein the length is between 20 and 80 atoms.

Claims

1. A polymer hydrogel comprising: a) a polymer formed by the crosslinking reaction of a polymeric unit A according to formula (I), ##STR00018## with a crosslinking unit B according to formula (II) ##STR00019## b) and water, wherein n=100-10,000; m=independently; FG is a functional moiety that can be covalently coupled to the complementary functional moiety F1 or F2 of the crosslinking unit (B); wherein functional groups F1, F2, F and functional groups FG that can give covalent couplings are independently selected from alkyne-azide coupling, dibenzocyclooctyne-azide coupling, bicyclo[6.1.0]non-4-yne-based-azide couplings, vinylsulphone-thiol coupling, maleimide-thiol coupling, methyl methacrylate-thiol coupling, ether coupling, thioether coupling, biotin-strepavidin coupling, amine-carboxylic acid resulting in amides linkages, alcohol-carboxylic acid coupling resulting in esters linkages, tetrazine-trans-cyclooctene coupling and NHS-ester (N-hydroxysuccinimide ester)-amine coupling; k=0.01-0.05; h=0, 1 or 2; the spacer is an organic moiety, having a main chain comprising at least two functional moieties F1 and F2, wherein the length of the crosslinker in the extended conformation as determined by molecular modeling (including the spacer and the functional groups F1 and F2) is between 2.5 and 12 nm, or wherein the length is between 20 and 80 atoms.

2. The polymer hydrogel according to claim 1, wherein the amount of the polymer in the hydrogel ranges between 0.01 wt. % and 1 wt %, wherein the amount of water in the hydrogel ranges between 90 and 99.99 wt. % relative to the total weight of the hydrogel.

3. The polymer hydrogel according to claim 1, wherein the concentration of the functional groups FG ranges between 20-200 M.

4. The polymer hydrogel according to claim 1, wherein the molar ratio between the functional group FG and the functional groups F1, F2, and F ranges between 0.5:1 and 2:1.

5. The polymer hydrogel according to claim 1, wherein each m is 3 or 4.

6. The polymer hydrogel according to claim 1, wherein k ranges between 0.02 and 0.04.

7. The polymer hydrogel according to claim 1, wherein the couplings are based on azide-alkene and/or azide-alkyn coupling.

8. The polymer hydrogel according to claim 1, wherein the functional groups F1 and F2 of the crosslinking unit are selected from ##STR00020## and their derivatives.

9. The polymer hydrogel according to claim 1, wherein the spacer comprises polyethylene glycol units, having from 3 to 26 ethylene glycol units.

10. The polymer hydrogel according to claim 1, wherein the spacer comprises a short peptide sequence, that is a cleavable metalloproteinase (MMP), VPMSMRG, VPMSMRGG, RPMSMR, IPESLRAG, IPVSLRSG, VPLSLYSG, VPMSMR, PAYYTA, GPQGIWGQ, SGESPAYYTA, GPQGIAGQ, RPFSMIMG, VPLSLTMG, YAAPVRGG, GTAGLIGQ or GDQGIAGF, wherein V=Valine; P=Proline; M=Methionine; S=Serine; R=Arginine; G=Glycine; I=Isoleucine; E=Glutamic Acid; L=Leucine; A=Alanine; Y=Tyrosine; Q=Glutamine; K=lysine and F=Phenylalanine.

11. The polymer hydrogel according to claim 1, wherein the crosslinking unit B is defined by any one of formulas IX-XII ##STR00021## wherein is a photocleavabe dye that, as a result of the photochemical reaction, cleaves the spacer.

12. The polymer hydrogel according to claim 1, wherein the hydrogel has a R value of at least 40%, wherein R is defined as the percentage of stiffness remaining at 5 C., G.sub.5, as compared to the stiffness at the 37 C., G.sub.37:
R=100%G.sub.5/G.sub.37.

13. A process for preparing the polymer hydrogel according to claim 1, comprising the steps of: a. copolymerizing a first comonomer of an oligo(ethylene glycol) functionalized isocyanopeptide grafted with a functional group FG and a second comonomer of a non-grafted oligo(ethylene glycol) functionalized isocyanopeptide, wherein the molar ratio between the first comonomer and the second comonomer is 1:100 and 1:20 to prepare a polymer (A); b. dissolving polymer (A) in water to a concentration between 0.01 and 1 wt % polymer between 0 and 10 C. to prevent hydrogel formation; c. adding a crosslinking compound having a spacer unit and at least two functional groups F (=F1=F2=(F).sub.h) capable of reacting with the functional group FG, wherein the functional group FG and the functional group F are chosen to react and form a coupling, wherein the coupling is independently selected from the group consisting of alkyne-azide coupling, dibenzocyclooctyne-azide coupling, oxanorbornadiene-based-azide couplings, vinylsulphone-thiol coupling, maleimide-thiol coupling, methyl methacrylate-thiol coupling, ether coupling, thioether coupling, biotin-strepavidin coupling, amine-carboxylic acid resulting in amides linkages, alcohol-carboxylic acid coupling resulting in esters linkages, tetrazine-trans-cyclooctene and NHS-Ester (N-Hydroxysuccinimide ester)-amine coupling; d. bringing the hydrogel within 10 minutes to the desired crosslinking temperature for gel formation (fast) at temperatures above the LOST or gelation temperature and the reaction with a crosslinking compound (slower).

14. The process according to claim 13, wherein the temperature of reaction ranges between 20 and 50 C.

15. Cell cultures, comprising: cells, cell culture medium and the hydrogel according to claim 1.

16. The polymer hydrogel according to claim 11, wherein the number of ethylene glycol units (m) ranges independently between 2 and 10.

17. The polymer hydrogel according to claim 16, wherein k ranges between 0.02 and 0.04.

18. The polymer hydrogel according to claim 17, wherein the amount of polymer in the hydrogel ranges between 0.01 wt. % and 1 wt. %, wherein the amount of water in the hydrogel ranges between 90 and 99.99 wt. % relative to the total of the hydrogel.

19. The polymer hydrogel according to claim 11, wherein the concentration of functional groups FG ([FG]) ranges between 20-200 M.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the schematic formation of crosslinked PIC hydrogel.

(2) FIG. 2 shows the effect of formation of intra-bundular crosslinks at higher temperatures on the mechanical properties after cooling the crosslinked polymer hydrogel.

(3) FIG. 3 shows the effect of crosslinking at different temperatures on the properties of the hydrogels as a function of temperature.

(4) FIG. 4 shows the effect of using short (EO4) relative to long (EO26) spacers present in the crosslinking unit on the storage modulus.

(5) FIG. 5 shows the effect of using short (EO4) relative to long (EO26) spacers present in the crosslinking unit on the differential modulus.

(6) FIG. 6 shows the thermal stability of the gel as a function of the crosslink concentration [crossl] given in M. The stability is quantified by R=100%G.sub.5/G.sub.37.

DETAILED DESCRIPTION OF THE INVENTION

Experimental

(7) Materials: Toluene was distilled over sodium. Water was purified with a MilliPore MilliQ system, (mQ water 18.2 M). Unless stated otherwise, all chemicals were obtained from commercial sources and used without further purification. If no further details are given the reaction was performed under ambient atmosphere and temperature. Column chromatography was performed using silica gel (0.060-0.200 mm) provided by Baker.

Synthesis of the Azide-Functionalized Isocyanide Ionomer

(8) The azide-functionalized isocyanide monomer was synthesized following a modified literature procedure. The first and last step of the procedure were altered.

Synthesis of Tetraethylene Glycol p-Toluenesulfonate

(9) Tetraethylene glycol (52.68 g, 271 mmol) was dissolved in 10 mL THF and the solution was cooled to 0 C. A solution of NaOH (1.81 g, 45.25 mmol) in 10 mL water was added and the mixture was stirred vigorously for 5 minutes. A solution of tosyl chloride (8.08 g, 42.4 mmol) in 70 mL THF was added drop wise. The reaction mixture was stirred for 2.5 h. Subsequently, the solution was poured onto ice water (200 mL) and 50 mL of dichloromethane (DCM) was added. The water layer was extracted 4 times with 100 mL of DCM. The combined organic layers were dried using anhydrous NaSO.sub.4 and the solvents were removed in vacuo. The product was purified using flash chromatography (SiO.sub.2, EtOAc) to yield 11.8525 g (80%) of a yellow oil.

(10) Analysis: FT-IR (cm.sup.1, ATR) 3442 (OH), 2870 (CH), 1597 (NH), 1453 (CH), 1352 (SO), 1175 (SO), 1096 (CO); .sup.1H-NMR .sub.H (300 MHz; CDCl.sub.3; Me.sub.4Si) 7.80 (dd, J=7.81 Hz, 2H, CH.sub.Ar), 7.33 (d, J=7.35 Hz, 2H, CH.sub.ArS), 4.17 (m, 2H, OCH.sub.2CH.sub.2), 3.65 (m, 16H, CH.sub.2), 2.45 (s, 3H, CH.sub.3); .sup.13C-NMR .sub.C (75 MHz; CDCl.sub.3; Me.sub.4Si) 21.16 (1C, CCH.sub.3), 61.0 (1C, COH), 68.13 (1C, COS), 69.0 (1C, OCH.sub.2), 70.0, 70.1, 70.1, 70.2 (4C, OCH.sub.2), 70.8, 72.0 (2C, OCH.sub.2), 127.5 (2C, CHCCH), 129.5 (2C, CHCCH), 139.7 (1C, CCH.sub.3), 144.5 (1C, CHCS).

Synthesis of (R)-2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl 2-((S)-2-isocyanopropanamido)propanoate

(11) (R)-2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl 2-((S)-2-formamidopropanamido)propanoate (640 mg, 1.64 mmol) was dissolved in 60 mL of freshly distilled DCM. To this solution, methyl N-(triethylammoniumsulfonyl)carbamate (Burgess reagent; 594 mg, 2.49 mmol) was added. The mixture was stirred at rt for 6 h until no starting material was observed on TLC. The solvents were removed in vacuo, and the product was purified using flash chromatography (SiO.sub.2, 3:1 DCM:MeCN) to yield 0.436 g (72%) of a pale yellow oil.

(12) Analysis: FT-IR (cm.sup.1, ATR) 3318 (NH), 2875 (CH), 2142 (CN), 2105 (N.sub.3), 1744 (CO), 1540 (NH), 1453 (CH), 1123 (CO); .sup.1H-NMR .sub.H (300 Hz; CDCl.sub.3; Me.sub.4Si) 7.00 (bd, 1H, NH), 4.59 (m, 1H, NHCH(CH.sub.3)C(O)O), 4.32 (m, 3H, (C(O)OCH.sub.2CH.sub.2O), CNCH(CH3)C(O)NH), 3.67 (m, 12H, (OCH.sub.2CH.sub.2).sub.3), 3.39 (m, 2H, N.sub.3CH.sub.2), 1.65 (d, J=7.2, 3H, CNCH(CH.sub.3)C(O)), 1.48 (d, J=7.2, 3H, CNCH(CH.sub.3)C(O)); .sup.13C NMR .sub.C (75 MHz; CDCl.sub.3; Me.sub.4Si) 170.69 (1C, CH(CH.sub.3)C(O)OCH.sub.2), 165.72 (1C, CH(CH.sub.3)C(O)NH), 70.69, 70.65, 70.61, 70.56, 70.02, 68.81 (1C, CH.sub.2CH.sub.2O), 50.66 (1C, CH.sub.2N.sub.3), 48.56 (CNCH), 19.66, 18.04 (1C, CH(CH3)CO); S (ESI) m/z [M+Na].sup.+ (C.sub.15H.sub.25N.sub.5O.sub.6Na), calcd 394.17; found 394.1.

Polymerization

(13) The commercial methoxy-terminated isocyanide monomer was further purified by flash chromatography (SiO.sub.2, 1:3 MeCN:DCM) before use. The purified monomer (250 mg, 0.79 mmol) and the azide functionalized isocyanide monomer (10.1 mg, 0.027 mmol) were dissolved in freshly distilled toluene (4 mL). Subsequently, 81.7 L of a freshly prepared solution of 1 mM Ni(ClO.sub.4).sub.2.6H.sub.2O in freshly distilled toluene containing 10% EtOH was diluted to 1 mL using freshly distilled toluene. This catalyst dilution was added to the monomer solution. The resulting mixture was stirred for 72 h at rt. Afterwards, the reaction mixture was diluted with dichloromethane and the product was precipitated in di-isopropyl ether. This workup was repeated thrice. The product was yielded as 243.6 mg (94%) of a yellow solid.

(14) FT-IR v.sub.max film (cm.sup.1): 3268, 2876, 1742, 1657, 1532, 1455, 1264, 1217, 1065, 729, 703. M.sub.v=599 kDa, UV-Vis .sub.max (25 C., milliQ)=245 nm.

Synthesis of Long Crosslinker

(15) 4-Dimethylaminopyridine (DMAP, 0.5 mg, 4.4 mol), DBCO-amine (38.1 mg, 138 mol) and PEG(1000) bis-acetic acid (49.7 mg, 44 mol) were dissolved in freshly distilled DCM (5 mL).

(16) ##STR00017##

(17) The reaction mixture was cooled to 0 C. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 28 mg, 146 mol) was added and the mixture was allowed to warm up to room temperature. The solution was stirred overnight at rt. Afterwards, the reaction mixture was diluted with DCM, and the organic layer was washed with water (350 mL) and brine (150 mL). The organic layer was dried with anhydrous Na.sub.2SO.sub.4 and the solvents were evaporated in vacuo. The product was dissolved in 1 mL of tetrahydrofuran (THF), filtered and the solvent was evaporated in vacuo. This procedure was repeated once. For further purification, the product was dissolved in a minimum amount of THF and precipitated in cold heptane. This procedure was repeated twice, after which the product was yielded as 59.7 mg (84%) of a yellow oil.

(18) .sup.1H-NMR (500 MHz, CDCl.sub.3, ppm) : 7.70 (d, 2H, C.sub.ArH), 7.46-7.31 (m, 14H, C.sub.ArH), 7.13 (br t, 2H, NH), 5.16 (d, 2H, C.sub.3H), 3.90 (q, 4H, C.sub.8H.sub.2), 3.80-3.60 (m, 88H, OCH.sub.2CH.sub.2, C.sub.3H), 3.60-3.49 (m, 8H, OCH.sub.2CH.sub.2), 3.44-3.32 (m, 4H, C.sub.6H.sub.2), 2.55 (dt, 2H, C.sub.5H), 2.02 (dt, 2H, C.sub.5H). .sup.13C-NMR (126 MHz, CDCl.sub.3, ppm) : 171.56 (C.sub.4), 169.79 (C.sub.7), 151.11 (C.sub.Ar), 148.07 (C.sub.Ar), 132.16 (C.sub.Ar), 129.09 (C.sub.Ar), 128.58 (C.sub.Ar), 128.29 (C.sub.Ar), 128.23 (C.sub.Ar), 127.73 (C.sub.Ar), 127.18 (C.sub.Ar), 125.53 (C.sub.Ar), 123.07 (C.sub.Ar), 122.55 (C.sub.Ar), 114.80 (C.sub.1), 107.74 (C.sub.2), 70.86 (CH.sub.2CH.sub.2O), 70.56 (CH.sub.2CH.sub.2O and C.sub.8), 70.27 (CH.sub.2CH.sub.2O), 55.45 (C.sub.3), 34.78 (C.sub.5, C.sub.6). MALDI-TOF: m/z=1377.6179, 1421.6465, 1465.6640, 1509.6913, 1553.7158, 1597.7458, 1641.7732, 1685.8016, 1729.8202, 1773.8499, 1817.8627, 1861.8839 and 1905.8782 which corresponds to DBCO-(CH.sub.2CH.sub.2O).sub.n-DBCO+Na.sup.+ (n=14-26) and 1569.6882, 1613.7091, 1657.7335 and 1701.7500 which corresponds to DBCO-(CH.sub.2CH.sub.2O).sub.n-DBCO+K.sup.+ (n=17-20). FT-IR v.sub.max film (cm.sup.1): 3507 (NH), 2870 (CH), 1655 (CO amide), 1535, 1466, 1449, 1398, 1325, 1250, 1206, 1101 (CN), 949, 846, 729. UV-Vis .sub.max (25 C., milliQ)=290.5 nm. .sub.290.5 (milliQ)=16430 L mol.sup.1.Math.cm.sup.1.

Crosslinking Reaction

(19) When Dibenzocyclooctyn (DBCO)-based crosslinkers are used, the conversion of the DBCO groups can be followed experimentally using UV-vis spectrosopy. To this end, the (decreasing) DBCO absorbance at =304 nm is traced during the crosslinking reaction. The concentration remaining DBCO groups can be calculated from the extinction coeffcient at =304 nm: .sub.304=7610 L mol.sup.1 cm.sup.1.

Preparation of the Hydrogel from the Copolymer

(20) A homogeneous solution of 2 mg mL.sup.1 polyisocyanide copolymer was prepared by dissolving the dry polymer in the appropriate amount of milliQ water over night at 4 C., and occasionally shaking. All polymer solutions were stored at 20 C. until further use.

Example 1

Preparing a Crosslinked Hydrogel

(21) A solution of 2 mg mL.sup.1 of the commercially available short crosslinker in DMSO, or 2 mg mL.sup.1 of the long crosslinker in milliQ water, was prepared and diluted to the desired concentration using milliQ water. The concentrations were chosen such that the polymer and crosslinker solutions could be mixed in a 1:1 ratio to achieve the ratio [DBCO]/[N.sub.3]=1. Before measurement, the solution of the polyisocyanide copolymer and a solution of the long crosslinker or the short crosslinker were mixed on ice in a 1:1 ratio, briefly homogenized and immediately heated to 37 C., for 1 hour, in a TA Instruments Discovery Hybrid Rheometer. After this, the gel was cooled down to 5 C. and was allowed to stabilize for 10 minutes. A gel with a storage modulus of 161 Pa was obtained at 37 C. The storage modulus at 5 C. was 95 Pa. The conversion of DBCO after 15 minutes was 8110% (measured using UV-Vis). R=59%. Crosslink density=34 M.

Example 2

Stability of the Crosslinked Hydrogel

(22) Example 1 was repeated except that the samples were allowed to stabilize for 19 hours instead of 10 minutes at 5 C. The storage modulus of this sample after 10 minutes at 5 C. was 103 Pa (R=63%), and the storage modulus after 19 hours was 101 Pa (R=64%, Crosslink density=32 M).

Experiment 3

Low Crosslinker Concentration Gives Loose Bundles

(23) Example 1 was repeated except that the ratio [DBCO]/[N.sub.3] was 0.5 instead of 1.0. The storage modulus of this gel at 37 C. was 185 Pa, and the storage modulus at 5 C. was 55 Pa. The conversion of DBCO after 15 minutes was 9410% (measured using UV-Vis). R=30% Crosslink density=12 M.

Experiment 4

High Crosslinker Concentration Gives Loose Bundles

(24) Example 1 was repeated except that the ratio [DBCO]/[N.sub.3] was 2.0 instead of 1.0. The storage modulus of the gel at 37 C. was 156 Pa, and the storage modulus at 5 C. was 31 Pa. The conversion of DBCO after 15 minutes was 6310% (measured using UV-Vis). R=20%, Crosslink density=13 M.

Example 5

Higher Crosslinking Temperature

(25) Example 1 was repeated except that the crosslinking temperature was 50 C. instead of 37 C. The storage modulus of the gel at 50 C. was 478 Pa, and the storage modulus at 5 C. was 218 Pa. R=46%. Crosslink density not experimentally determined. Estimated (considering 90 reaction yield): 34 M.

Example 6

Lower Crosslinking Temperature

(26) Example 1 was repeated except that the crosslinking temperature was 25 C. instead of 37 C. The storage modulus of the gel at 25 C. was 48 Pa, and the storage modulus at 5 C. was 31 Pa. R=65%. Crosslink density not experimentally determined. Estimated (considering 90 reaction yield): 34 M.

Experiment 7

Longer Crosslinker

(27) Example 1 was repeated except that the long crosslinker, dissolved as 2 mg mL.sup.1 in milliQ, was used (6-10 nm instead of 3 nm). The storage modulus of the gel at 37 C. was 125 Pa, and the storage modulus at 5 C. was 40 Pa. The conversion of DBCO after 15 minutes was 8910% (measured using UV-Vis). R=32%. Crosslink density not experimentally determined. Estimated (considering 90 reaction yield): 34 M.

Experiment 8

Too Low Crosslinker Concentration Disintegrated Gel

(28) Example 1 was repeated except that the ratio [DBCO]/[N.sub.3] was 0.1 instead of 1.0. The storage modulus of the gel at 37 C. was 155 Pa, and the storage modulus at 5 C. was 2 Pa. The concentration of DBCO was too low to measure conversion using UV-Vis measurements. R<1%, Crosslink density=0.3 M.

Experiment 9

Too High Crosslinker Concentration Disintegrated Gel

(29) Example 1 was repeated except that the ratio [DBCO]/[N.sub.3] was 10 instead of 1.0. The storage modulus of the gel at 37 C. was 260 Pa, and the storage modulus at 5 C. was 0.3 Pa. The concentration of DBCO was too low to measure conversion using UV-Vis measurements. R<1%, Crosslink density=0.3 M.

Experiment 10

No Crosslinker

(30) Example 1 was repeated except that no crosslinker was used. The storage modulus of the gel at 37 C. was 160 Pa, and the storage modulus at 5 C. was 0.6 Pa. R=0.4%. Crosslink density=0 M.

Experiment 11

Random Crosslinking of the PIC Polymers, Not in the Bundles

(31) Example 1 was repeated, except that the hydrogel was not heated 37 C. after homogenisation of the components, but kept at 5 C. to allow the crosslinking reaction to complete. After 2 hrs, the modulus stabilised at 7 Pa, indicative of a very weak gel. Crosslink density (but not in the bundles) estimated at 34 M.

Experiment 12

Random Crosslinking of the PIC Polymers, Not in the Bundles

(32) Example 1 was repeated, but in the presence of 1 M NaI, which increases the LCST of the PIC polymer. After homogenisation of the components, the sample was heated to 25 C. (below the LCST) to allow the crosslinking reaction to complete. After 2 hrs, the modulus stabilised at below 1 Pa, indicative of a very weak gel. Crosslink density (but not in the bundles) estimated at 34 M.