Abstract
Block copolymers have a general chemical structure of one of the formulas [A].sub.n-[B].sub.m and [B].sub.n-[A].sub.m, wherein block [A] is a poly(2-oxazine) and wherein block [B] is a poly(2-oxazoline). The block copolymers have desired thermogelling and rheological properties and are useful as carrier materials for active ingredients such as drugs, cells, proteins, and other active ingredients.
Claims
1. A thermoresponsive hydrogel which comprises an aqueous solution of a block copolymer having a general chemical structure of one of the following formulas:
[A].sub.n-[B].sub.m and [B].sub.n-[A].sub.m wherein block [A] of the block copolymer is chosen from the group consisting of 2-n-propyl-2-oxazine, 2-cyclopropyl-2-oxazine, and 2-butyl-2-oxazine, wherein block [B] of the block copolymer is chosen from the group consisting of 2-methyl-2-oxazoline and 2-ethyl-2-oxazoline, wherein n is in the range of 20 to 300, wherein m is in the range of 20 to 300, and wherein n and m have the same or approximately the same value.
2. The thermoresponsive hydrogel according to claim 1, wherein the block copolymer is prepared by a two-stage co-polymerization of 2-oxazine and 2-oxazoline.
3. The thermoresponsive hydrogel according to claim 1, wherein the block copolymer has a general chemical structure of one of the following formulas: ##STR00008## wherein R is an alkyl group and R.sub.1 is a piperidine group, wherein n is in the range of 20 to 300, wherein m is in the range of 20 to 300, and wherein n and m have the same or approximately the same value.
4. The thermoresponsive hydrogel according to claim 1, wherein the block copolymer has a general chemical structure of the formula: ##STR00009## wherein n and m each have a value of 50.
5. The thermoresponsive hydrogel according to claim 1, wherein the block copolymer has a general chemical structure of the following formula: ##STR00010## wherein n and m each have a value of 100.
6. The thermoresponsive hydrogel according to claim 1, wherein the block copolymer gels at a temperature above 10° C.
7. The thermoresponsive hydrogel according to claim 6, wherein the block copolymer gels at a temperature above 25° C.
8. The thermoresponsive hydrogel according to claim 7, wherein the block copolymer gels at a temperature above 30° C.
9. The thermoresponsive hydrogel according to claim 8, wherein the block copolymer gels at a temperature above 35° C.
10. A composition which comprises an active ingredient and a thermoresponsive hydrogel according to claim 1 as a carrier material for the active agent.
11. The composition according to claim 10, wherein the active agent is embedded in the carrier material.
12. The composition according to claim 11, wherein the carrier material is characterized by time-delayed release or distribution of the active ingredient.
13. A drug delivery system which comprises a drug or drugs to be delivered and a thermoresponsive hydrogel according to claim 1 as a carrier material for the drug or drugs to be delivered.
14. The drug delivery system of claim 13, wherein the carrier material is characterized by time-delayed release or distribution of the drug or drugs.
15. A composition which comprises cells and a thermoresponsive hydrogel according to claim 1 as a carrier material for the cells.
16. A composition which comprises proteins and a thermoresponsive hydrogel according to claim 1 as a carrier material for the proteins.
17. A block copolymer which has a general chemical structure of the formula: ##STR00011## wherein n and m each have a value of 50.
18. A block copolymer which has a general chemical structure of the following formula: ##STR00012## wherein n and m each have a value of 100.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0072] FIG. 1 shows GPC traces for different batches of Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester;
[0073] FIG. 2 shows the temperature dependent rheology analysis with storage modulus (G′) and loss modulus (G″) for 20 wt.-% of Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester;
[0074] FIG. 3 shows an .sup.1H-NMR spectra of batch P1 in methanol-d.sup.4 at 298 K of Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester;
[0075] FIG. 4 shows an .sup.1H-NMR spectra of batch P2 in methanol-d.sup.4 at 298 K of Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester;
[0076] FIG. 5 shows an .sup.1H-NMR spectra of batch P3 in methanol-d.sup.4 at 298 K of Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester;
[0077] FIG. 6 shows an .sup.1H-NMR spectra of batch P4 in methanol-d.sup.4 at 298 K of Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester;
[0078] FIG. 7 shows a .sup.1H-NMR spectra of batch P5 in methanol-d.sup.4 at 298 of Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester;
[0079] FIG. 8 shows an amplitude sweep used to determine the LVE-range at 10° C. and 50° C. for batch P2;
[0080] FIG. 9 shows a flow curve at 37° C. for 20 wt.-% of Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester;
[0081] FIG. 10 shows shear recovery at 37° C. for 20 wt.-% of Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester;
[0082] FIG. 11 shows the temperature- and concentration-dependent viscosity of Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester at different concentrations compared with the temperature- and concentration-dependent viscosity of the Poloaxamer F127 at 10 wt.-%;
[0083] FIG. 12 shows temperature-dependent SANS scattering data of Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester at 20 wt.-%;
[0084] FIG. 13 shows representative fits for a bi-continuous sponge like structure for lowest temperature (at 6.9° C.) and highest temperature (39.7° C.) of Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester at 20 wt.-%;
[0085] FIG. 14 shows resulting correlation length ξ and characteristic domain size d of Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester at 20 wt.-%;
[0086] FIG. 15 shows fits of selected datasets over the complete temperature range of Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester at 20 wt.-%;
[0087] FIG. 16 shows the proliferation of NIH-3T3 cells in the presence of various polymer concentrations of Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester as analyzed by WST-1 assay at 20 wt.-%;
[0088] FIG. 17 shows Cell viability of NIH 3T3 fibroblasts;
[0089] FIG. 18 shows a flow cytometry analysis of Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester at 20 wt.-%;
[0090] FIG. 19 shows a flow cytometry analysis of NIH 3T3 fibroblasts cultivated in Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester at 20 wt.-%;
[0091] FIG. 20 shows a temperature-dependent rheological analysis of Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester (P2) at a concentration of 20 wt.-%;
[0092] FIG. 21 shows a phase diagram of aqueous solutions of Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester (P2) dependent on concentration and temperature;
[0093] FIG. 22 shows the temperature dependent rheology with storage modulus (G′) and loss modulus (G″) for 20 wt.-% of Methyl-P[nPrOzi.sub.100-b-MeOx.sub.100]-piperidine-4-carboxylic acid ethyl ester and Methyl-P[nPrOzi.sub.100-b-MeOx.sub.100]-tert-butyl-piperazine-1-carboxylat;
[0094] FIG. 23 shows the temperature dependent rheology with storage modulus (G′) and loss modulus (G″) for 20 wt.-% of Propynyl-P[nPrOzi.sub.100-b-MeOx.sub.100]-piperidine-4-carboxylic acid ethyl ester, Propynyl-P[nPrOzi.sub.100-b-MeOx.sub.10]-methyl 3-mercaptopropionate and Propynyl-P[nPrOzi.sub.100-b-MeOx.sub.100]-hydroxy;
[0095] FIG. 24 shows GPC traces of Methyl-P[nPrOzi.sub.100-b-MeOx.sub.100]-piperidine-4-carboxylic acid ethyl ester and Methyl-P[nPrOzi.sub.100-b-MeOx.sub.100]-tert-butyl-piperazine-1-carboxylat:
[0096] FIG. 25 shows GPC traces of Propynyl-P[nPrOzi.sub.100-b-MeOx.sub.100]-piperidine-4-carboxylic acid ethyl ester, Propynyl-P[nPrOzi.sub.100-b-MeOx.sub.100]-methyl 3-mercaptopropionate and Propynyl-P[nPrOzi.sub.100-b-MeOx.sub.100]-hydroxy;
[0097] FIG. 26 shows an .sup.1H-NMR spectra of Methyl-P[nPrOzi.sub.100-b-MeOx.sub.100]-piperidine-4-carboxylic acid ethyl ester in methanol-d.sup.4 at 298 K;
[0098] FIG. 27 shows an .sup.1H-NMR spectra of Methyl-P[nPrOzi.sub.100-b-MeOx.sub.100]-tert-butyl-piperazine-1-carboxylat in methanol-d.sup.4 at 298 K;
[0099] FIG. 28 shows an .sup.1H-NMR spectra of Propynyl-P[nPrOzi.sub.100-b-MeOx.sub.100]-piperidine-4-carboxylic acid ethyl ester in methanol-d.sup.4 at 298 K;
[0100] FIG. 29 shows an 1H-NMR spectra of Propynyl-P[nPrOzi.sub.100-b-MeOx.sub.1]-methyl 3-mercaptopropionate in methanol-d.sup.4 at 298 K;
[0101] FIG. 30 shows an 1H-NMR spectra of Propynyl-P[nPrOzi.sub.100-b-MeOx.sub.10]-hydroxy in methanol-d.sup.4 at 298 K;
[0102] FIG. 31 shows the temperature dependent rheology with storage modulus (G′) and loss modulus (G″) for 20 wt.-% of Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-tert-butyl-piperazine-1-carboxylat, contaminated with 10% Poly(n-butyl-2-oxazin);
[0103] FIG. 32 shows an .sup.1H-NMR spectra of Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-tert-butyl-piperazine-1-carboxylat, contaminated with 10% Poly(n-butyl-2-oxazin);
[0104] FIG. 33 shows a light microscope image of a printed constructs composed of orthogonal stacks of hydrogel strands with a base area of 12×12 mm.sup.2 and a strand-center to strand-center distance of 3 mm;
[0105] FIG. 34 shows cell loaded constructs; and
[0106] FIG. 35 shows results of FACS analysis on the influence of the printing process on the viability of NIH 3T3 fibroblasts.
DETAILED DESCRIPTION OF THE INVENTION
[0107] Reference will now be made in detail to the preferred embodiments of the present invention. It is to be understood that the following examples are illustrative only and the present invention is not limited thereto.
[0108] Several batches (P1 to P5) of Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester (poly(nPrOzi)-b-poly(MeOx) were analyzed using gel permeation chromatography (GPC) (FIG. 1), temperature dependent rheological properties (FIG. 2) and .sup.1H-NMR spectra (FIGS. 3 to 7). The results are first of all summarized in following Table 1:
TABLE-US-00001 TABLE 1 Degree of Polymerization (DP) and molar masses (kg/mol) obtained by NMR and GPC of batches P1 to P5. DP .sub.theo. DP.sub.exp.sup.[a] M.sub.n theo. Mn.sup.[b] Mw.sup.[b] ![custom-character]()
.sup.[b] P1 52/52 42/44 11.2 10.0 14.9 1.49 P2 50/50 57/55 10.8 7.3 8.5 1.17 P3 50/50 51/51 10.8 6.3 8.1 1.29 P4 50/58 55/50 11.5 6.4 8.2 1.28 P5 51/49 44/45 10.8 6.5 7.8 1.22 .sup.[a]Determined by end-group analysis (.sup.1H NMR spectroscopy in MeOD-d.sup.4 (300 MHz, 298K)); .sup.[b]Determined from GPC in DMF with LiBr (1 g/L) at 313K.
[0109] FIG. 1 shows the GPC traces for batches P1 to P5 of Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester, represented by curves 1, 3, 5, 7, 9. The elugrams obtained by GPC appear, with the exception of the first batch P1 (curve 1), nearly monomodal with only minor tailing to lower molar masses. Batch P1 exhibits a significant shoulder at higher molar masses, accordingly, dispersity is highest in this sample (![custom-character]()
=1.49).
[0110] FIG. 2 shows the temperature dependent rheology with storage modulus (G′) and loss modulus (G″) for 20 wt.-% of Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester. Storage modulus (G′) of batches P1 to P5 is represented by curves 11, 13, 15, 17 and 19 and loss modulus (G″) of batches P1 to P5 is represented by curves 21, 22, 23, 24, and 25.
[0111] The rheological properties of aqueous solutions of batch P1 (20 wt.-%, curves 11 and 21 in FIG. 2) were investigated dependent on temperature in the linear viscoelastic range. With regard to this, FIG. 8 shows the amplitude sweep used to determine the LVE-range at 10° C. and 50° C. for batch P2. Curves 26 (G″) and 27 (G′) show the results at 10° C. and curves 28 (G′) and 29 (G″) show the results at 50° C. A relatively sharp sol-gel transition at approximately 27° C. was observed. Notably, G″ starts to increase at much lower temperature than G′ (approximately 13° C.), which corresponds with the cloud point of PnPrOzi as homopolymer (M. M. Bloksma, R. M. Paulus, van Kuringen, Huub P C, F. van der Woerdt, H. M. L. Lambermont-Thijs, U. S. Schubert, R. Hoogenboom, Macromol. Rapid Commun. 2012, 33, 92-96).
[0112] After gelation, G′ reaches a plateau at about 4 kPa. Therefore, these gels are surprisingly strong compared to many other thermogelling polymers, for which values <1 kPa are more commonly found in the literature (C. Li, N. J. Buurma, I. Haq, C. Turner, S. P. Armes, V. Castelletto, I. W. Hamley, A. L. Lewis, Langmuir 2005, 21, 11026-11033; S. Xuan, C.-U. Lee, C. Chen, A. B. Doyle, Y. Zhang, L. Guo, V. T. John, D. Hayes, D. Zhang, Chem. Mater. 2016, 28, 727-737). A prominent exception are hydrogels of F127 at 20 wt.-% (approximately 10 kPa) (G. Grassi, A. Crevatin, R. Farra, G. Guamieri, A. Pascotto, B. Rehimers, R. Lapasin, M. Grassi, J. Colloid Interface Sci. 2006, 301, 282-290).
[0113] Comparing the different batches P1 to P5, it was found that for 20 wt.-% only batches P1 (curves 11 and 21), P2 (curves 13 and 22) and P5 (curves 19 and 25) formed such relatively strong gels (G″/G′=tan δ≈0.2). In contrast, P3 (curves 15 and 23) and P4 (curves 17 and 24) formed gels as evidenced by G′>G″, albeit very weak ones (tan δ≈1; G′<0.1 kPa). This was surprising as all batches, in particular batches P2 to P5, appeared very similar from GPC analysis (FIG. 1) and .sup.1H-NMR spectra (to be seen in FIGS. 3 to 7).
[0114] Batches P3 and P4 only show a somewhat more pronounced low-molecular tailing in the GPC elugrams (FIG. 1). Surprisingly, this minor tailing appears to influence the gelation behaviour very significantly, which emphasizes the problem of batch-to-batch reproducibility in the context of biomaterials research (R. Luxenhofer, Nanomedicine 2015, 10, 3109-3119). .sup.1H-NMR spectra shown in FIGS. 3 to 7 were in good agreement with the targeted polymer composition. Taking into account the requirements defined by Wang et al. batches P1, P2, and P5 appear suitable for bioprinting (S. Wang, J. M. Lee, W. Y. Yeong, Int. J. Bioprinting 2015).
[0115] In FIG. 9 a flow curve 31 at 37° C. for batch P2 (20 wt.-% of Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester) is shown. A crucial bioink parameter of batch P2 was investigated at 20 wt.-% and 37° C. (D. B. Kolesky, R. L. Truby, A. S. Gladman, T. A. Busbee, K. A. Homan, J. A. Lewis, Adv. Mater. 2014, 26, 3124-3130). The viscosity decreased from 4 kPa*s to 1 Pa*s with increasing shear rate from 0.01 s.sup.−1 to 100 s.sup.−1. Once the shear-stress stopped, the hydrogel regained strength very rapidly. This can be taken out of FIG. 10, which shows shear recovery at 37° C. for batch P2 (Storage Modulus G′, curve 33, Loss Modulus G″, curve 34). This combination of pronounced isothermal shear-thinning with rapid recovery is very desirable for bioinks.
[0116] For a better understanding of the rheological properties of the novel block copolymers, temperature and concentration dependent viscosity of aqueous solutions of P2 were measured (curve 35 (5 wt.-%), curve 37 (10 wt.-%), curve 41 (12.5 wt.-%), curve 43 (15 wt.-%), curve 45 (17.5 wt.-%), curve 47 (20 wt.-%), curve 49 (30 wt.-%) and comparison curve 51 F127 (10 wt.-%). The results can be seen in FIG. 11. It can be seen, that solutions of 20 wt.-% and more (curves 47 and 49 in FIG. 11) are gelling with increasing temperature. Below the LCST (lower critical solution temperature) of PnPrOzi a transparent solution of relatively low viscosity was observed at all concentrations.
[0117] Interestingly, at 5 wt.-% (curve 35) (and above LCST of nPrOzi), the solution became turbid (see also Phase diagram 115 in FIG. 21). The viscosity remained very low and decreased monotonously with temperature. In contrast, between approximately 10 and 20 wt.-% (curves 37 and 47) the solutions remained clear and liquid over the whole temperature range investigated (5 to 50° C.). The increase of viscosity started consistently around LCST, while the maximum of the viscosity goes through a plateau that shifts to higher temperatures with increasing polymer concentration.
[0118] At concentrations of 20 wt.-% and above, the solutions eventually gel. In this behavior the novel thermogelling polymers are quite distinct from F127 and P123, which also form gels at elevated temperature and/or concentration and are commonly used for gel plotting in biofabrication (N. E. Fedorovich, J. R. de Wijn, A. J. Verbout, J. Alblas, W. J. A. Dhert, Tissue Eng. Part A 2008, 14, 127-133). Important for the prospective use as injectable hydrogel or as bioink, the viscosity of the new material at low temperature is relatively low, in particular compared to the viscosity of Pluronic® block copolymers (compare 700 mPa*s (F127) vs. 7 mPa*s (P2) at 10 wt.-% and 10° C.). Even at 30 wt.-%, a solution of P2 at 10° C. (curve 49 in FIG. 11) has a lower viscosity (approx. 50%) than a 10 wt.-% solution of F127 (curve 51 in FIG. 11), which never forms a gel at this concentration. Based on these rheological experiments, it is possible to sketch a preliminary phase diagram for the new thermoresponsive materials.
[0119] This distinct rheological behavior is likely to be linked to the structure of polymer self-assemblies in water. For many, if not most thermogelling polymers the gelation is explained through an aggregation of spherical micelles into a cubic lattice. The novel hydrogel (batch P1, 20 wt.-%) was studied using small angle neutron scattering (SANS) at different temperatures (FIG. 12).
[0120] Instead, a model of a bi-continuous sponge-like structure as described by Teubner et al. was tested (M. Teubner, R. Strey, J. Chem. Phys. 1987, 87, 3195-3200). The expression they found is
[00001]
[0121] Here the proportionality constant C=(8π)/ξ![custom-character]()
η.sup.2![custom-character]()
c.sub.2V with ![custom-character]()
η.sup.2![custom-character]()
being the mean square fluctuation of the scattering density p and f is the correlation length c.sub.1 and c.sub.2 are given by
[00002]
wherein d is the characteristic domain size (periodicity). Ti model allows to fit the SANS data very well and yielded characteristic domain sizes and correlation lengths between 50 and 350 Å, depending on the temperature. This can be seen in FIG. 13, which shows the representative fits for a bi-continuous sponge-like structure for lowest temperature at 6.9° C. (curve 53) and highest temperature 39.7° C. (curve 61). For better visibility, the SANS scattering data at 39.7° C. (curve 61) and its respective fit was y-shifted using a factor of 64.
[0122] FIG. 14 shows the resulting correlation length ξ (curve 63) and characteristic domain size d (curve 65) of Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester. Important to note, the correlation length is a cutoff length, above which correlations are no longer noticeable in the system. As temperature increases, an increase in the domain size as well as in the correlation length was observed, the latter eventually exceeding the former (at approximately 18° C.).
[0123] As this is well below the gelation temperature, the correlation length apparently needs to exceed the characteristic distance considerably for a macroscopic rheological response from the system to occur. At temperatures just below 30° C., the increase of the correlation length levels off, which coincides with macroscopic gelation observed at ≈27° C. Both, SANS and rheology confirm that the structure of the novel hydrogels is very distinct from the commonly used Pluronic gels. This may open up new avenues for their use as biomaterials.
[0124] FIG. 15 shows the representative fits for a bi-continuous sponge like structure of selected datasets over the complete temperature range of Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester. For better visibility, SANS scattering data and their respective fits were shifted using a factor of 1 (6.9° C., curve 53), a factor of 4 (21.1° C., curve 55), a factor of 16 (30.0° C., curve 57), a factor of 64 (36.2° C., curve 59) and a factor of 256 (39.7° C., curve 61), respectively.
[0125] At non-gelling concentrations of up to 100 g/L no marked dose-dependent cytotoxicity in murine NIH 3T3 fibroblasts were found. The results can be taken out of bar chart 67 in FIG. 16 (bar 69: control sample, bar 71: blank sample, bar 73: 0.02 wt.-%, bar 75: 1 wt.-%, bar 77: 5 wt.-% and bar 79: 10 wt.-%). This is remarkable, as Schubert and co-workers found cytotoxicity below this concentration for POx homopolymers (M. Bauer, S. Schroeder, L. Tauhardt, K. Kempe, U. S. Schubert, D. Fischer, J. Polym. Sci., Part A: Polym. Chem. 2013, 51, 1816-1821.; M. Bauer, C. Lautenschlaeger, K. Kempe, L. Tauhardt, U. S. Schubert, D. Fischer, Macromol. Biosci. 2012, 12, 986-998).
[0126] At even higher concentration, the polymer undergoes gelation below 37° C., also in cell culture media. Therefore, cells were suspended in cell culture media supplemented with 25 wt.-% P2 and incubated for 24 h at 37° C. Also under these condition, the polymers/gels exhibited very good cytocompatibility. The results can be seen in FIGS. 17 to 19.
[0127] FIG. 17 shows the cell viability of NIH 3T3 fibroblasts as a bar chart 81. The bar chart 81 shows results for PI staining 83 with a bar 85 for the control sample, a bar 87 for the methanol treated sample and a bar 89 for the sample with cells in 25% of gel. As well, the bar chart 81 shows results for FDA staining 91 with a bar 93 for the control sample, a bar 95 for the methanol treated sample and a bar 97 for the sample with cells in 25% of gel.
[0128] FIG. 18 shows a flow cytometry analysis (FACS Calibur system) of NIH 3T3 fibroblasts cultivated in Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester, using a 488 nm laser with the emission channel FL2 (585 nm/±21 nm) for PI staining 83 (compare to FIG. 17) with curve 99 for control sample (see bar 85 in FIG. 17), curve 103 for the methanol treated sample (see bar 87 in FIG. 17) and curve 101 for the sample with cells in 25% of gel (see bar 89 in FIG. 17). A total number of 5000 events were counted with BD CellQuest™ Pro and the geometric mean fluorescence intensity was determined for each condition using Flowing Software (version 2.5.1; Turku Bioimaging).
[0129] FIG. 19 shows a flow cytometry analysis of NIH 3T3 fibroblasts cultivated in Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester for the emission channel FL1 (530 nm/±15 nm) for FDA staining 91 (compare to FIG. 17) with curve 105 for control sample (see bar 93 in FIG. 17), curve 107 for the methanol treated sample (see bar 95 in FIG. 17) and curve 109 for the sample with cells in 25% of gel (see bar 97 in FIG. 17).
[0130] In FIG. 20 a temperature-dependent rheological analysis of Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester at a concentration of 20 wt.-% (batch P2) is shown. Curve 111 shows the temperature-dependence of the storage modulus (G′), curve 113 shows the temperature-dependence of the loss modulus (G″). The gel point, determined at the intersection of G′ and G″, is located at 35° C., so just below body temperature. The gel point moves to lower temperatures with increasing the molar mass. The resulting hydrogels are quite soft with a loss modulus (G′) of around 4 kPa. In contrast to the gelling temperature, the storage modulus (G′) seems to be independent of the molar mass.
[0131] FIG. 21 shows a phase diagram 115 of aqueous solutions of Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester dependent on concentration and temperature. Increasing temperature, at concentrations below 10 wt.-% muddy solutions are obtained, which can be explained by nano- and microscaling aggregation.
[0132] Further, several batches (P1a to P6a) of R—P[nPrOzi.sub.100-b-MeOx.sub.100]-R.sup.1 were analyzed using gel permeation chromatography (GPC) (FIGS. 24 and 25), temperature dependent rheological properties (FIGS. 22, 23 and 31) and .sup.1H-NMR spectra (FIGS. 26 to 30 and 32). The results are first summarized in following Table 2.
TABLE-US-00002 TABLE 2 Degree of Polymerization (DP) and molar masses (kg/mol) obtained by NMR and GPC of batches P1a to P6a. DP .sub.theo. DP.sub.exp.sup.[a] M.sub.n theo. M.sub.n Mw ![custom-character]()
P1a 99/100 94/94 21.3 7.0.sup.[c] 9.4.sup.[c] 1.34.sup.[c] P2a 102/104 110/104 22.0 8.1.sup.[c] 11.5.sup.[c] 1.42.sup.[c] P3a 100/100 100/99 21.4 12.3.sup.[b] 19.2.sup.[b] 1.56.sup.[b] P4a 100/100 100/97 21.4 14.7.sup.[b] 21.2.sup.[b] 1.44.sup.[b] P5a 100/100 99/95 21.3 15.9.sup.[b] 20.8.sup.[b] 1.31.sup.[b] P6a 50/45/5 55/50/6 10.9 6.4.sup.[c] 7.4.sup.[c] 1.15.sup.[c] .sup.[a]Determined by end-group analysis (.sup.1H NMR spectroscopy in MeOD-d.sup.4 (300 MHz, 298K)); .sup.[b]Determined from GPC in DMF with LiBr (1 g/L) at 313K; .sup.[c]Determined form GPC in HFIP with potassium triflate (3g/L) at 313K.
[0133] FIG. 22 shows the temperature dependent rheology with storage modulus (G′) and loss modulus (G″) for 20 wt.-% of Methyl-P[nPrOzi.sub.100-b-MeOx.sub.100]-piperidine-4-carboxylic acid ethyl ester (batch P1a) and Methyl-P[nPrOzi.sub.100-b-MeOx.sub.100]-tert-butyl-piperazine-1-carboxylate (batch P2a). Storage modulus (G′) and loss modulus (G″) of batch P1a are represented by curves 117, 119 and storage modulus (G′) and loss modulus (G″) of batch P2a are represented by curves 121, 123. It can be seen, that the use of 1-BOC Piperazine (curves 121, 123) instead of ethyl-4-piperidinecarboxylate (curves 117, 119) as a termination molecule does no influence the gelation behavior of the block copolymer. The storage modulus G′ as well as loss modulus (G″) remain unchanged.
[0134] Same can be taken out of FIG. 23. FIG. 23 shows the temperature dependent rheology with storage modulus (G′) and loss modulus (G″) for 20 wt.-% of Propynyl-P[nPrOzi.sub.100-b-MeOx.sub.100]-piperidine-4-carboxylic acid ethyl ester (batch P3a), Propynyl-P[nPrOzi.sub.100-b-MeOx.sub.100]-methyl 3-mercaptopropionate (batch 4a) and Propynyl-P[nPrOzi.sub.100-b-MeOx.sub.100]-hydroxy (batch 5a). Storage modulus (G′) and loss modulus (G″) of batch P3a are represented by curves 125, 127, storage modulus (G′) and loss modulus (G″) of batch P4a are represented by curves 129, 131 and storage modulus (G′) and loss modulus (G″) of batch P5a are represented by curves 133, 135.
[0135] The results shown in FIG. 23 also suggest, that the observed effects occur independently of the end groups. By using different N-, O- and S-nucleophiles as termination molecules, the results shown in FIG. 22 could be verified showing the broad range of possible termination molecules used for synthesis.
[0136] FIG. 24 shows the GPC traces of batch P1a (curve 137) and batch P2a (curve 139) measured in HFIP. The GPC elugrammes 137, 139 have a moderately narrow molecular weight distribution ((Ð<1.5) and also show no differences as a result of the used termination molecule.
[0137] FIG. 25 shows the GPC traces of batch P3a (curve 141), batch P4a (curve 143) and batch P5a (curve 145) measured in DMF. These polymers were used for the preparation of the gels, which are characterized by the temperature-sweep shown in FIG. 23.
[0138] FIGS. 26 to 30 show the 1H-NMR spectra of batches P1a (FIG. 26), P2a (FIG. 27), P3a (FIG. 28), P4a (FIG. 29) and P5a (FIG. 30) in methanol-d.sup.4 at 298 K each. The 1H-NMR spectra 26, 27, 28, 29, 30 of all block copolymers show that there are no detectable rests of solvent, which may influence the gelling process (the formation of the gel) of the respective block copolymer. In addition, the desired degree of polymerization could be determined by endgroup analysis.
[0139] FIG. 31 shows the temperature dependent rheology with storage modulus (G′) and loss modulus (G″) for 20 wt.-% of contaminated Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-tert-butyl-piperazine-1-carboxylat (batch P6a). The storage modulus (G′) is represented by curve 147 and the loss modulus (G″) is represented by curve 149. FIG. 32 shows an .sup.1H-NMR spectra of batch P6a.
[0140] In detail, the Poly(2-n-propyl-2-oxazin)-block was deliberately contaminated with 10% Poly(n-butyl-2-oxazin). The resulting block copolymers are opaque, but exhibit storage modulus (G′) that is increased by a factor of 2 (curve 147). As a result, the material can store more deformation energy and influences the effect of reverse deformation. Due to the more pronounced elastic character, a higher degree of cross-linking can be assumed. In this context, it must be taken into account, that it is exclusively a reversible physical cross-linking reaction.
[0141] FIG. 33 shows a light microscope image of a printed constructs composed of orthogonal stacks of hydrogel strands. To be able to work at room temperature without risking ink liquefaction, experiments were conducted using a 20 wt.-% concentration of batch P2a. The pronounced shear thinning of the material enabled processing it at room temperature with a pressure of 1.2 bars using 0.25 mm inner diameter needles. With these settings, it was possible to generate defined constructs composed of orthogonal stacks of hydrogel strands with a base area of 12×12 mm.sup.2 and a strand-center to strand-center distance of 3 mm.
[0142] Furthermore, as can be seen in FIG. 34, by mixing 1.0 million NIH-3T3 fibroblasts into this ink cell loaded constructs could be generated. The cells did not influence the printability of the material and the same setting as for cell-free inks could be applied to process the bioinks.
[0143] The cell distribution within the constructs was homogeneous throughout the entire constructs. The homogenous cell distribution was facilitated due to the thermoresponsive properties of the material. At low temperatures (ice bath) the ink has a very low viscosity and cells are readily distributed within the material via repeated mixing by pipetting. Once taken of the ice, the immediate, temperature driven viscosity increase preserved the homogenous cell distribution within the ink until the material was dispensed. As noted by Malda et al., it can be challenging to homogeneously distribute cells in highly viscous bioinks due to various issues (air bubbles, difficult pipetting/handling) (V. H. M. Mouser, F. P. W. Melchels, J. Visser, W. J. A. Dhert, D. Gawlitta, J. Malda, Biofabrication 2016, 8, 35003).
[0144] To analyze if dispensing had a negative effect on cell viability, NIH-3T3 cells included into biofabricated scaffolds were further investigated via flow cytometry. The results can be taken out of FIG. 35. While the untreated control represents cells in medium, the control represents cells that were dispersed in the bioink but not printed. This revealed similar levels of cytocompatibility (91.5%±0.8%—bar 159) compared to cells incorporated into the material without further processing (92.8%±1.7%—bar 161) and untreated control cells (98.9%±0.18%—bar 163) Therefore, the printing process seems to have no effect on the cell viability when using our bioink.
[0145] In summary, new thermogelling synthetic block copolymers are presented, comprising a hydrophilic block [A] or [B] and a thermoresponsive block [A] or [B], which are an excellent bioink candidates. The new gels are optical transparent and have a very suitable and adjustable gelling temperature. The synthesis of the polymers is easy and to be scaled well. The gelation process of all describes molecules is very fast. The combination of thermogelation, excellent biocompatibility and isothermal shear-thinning is particularly attractive for many applications including drug delivery, biofabrication, cell culture or tissue engineering. The particularities of the rheological properties can be conveniently fine-tuned via the polymer composition while the chemical functionalization via chain termini can be realized without having an impeding influence on the desirable rheological properties.
[0146] The following is a summary list of reference numerals and the corresponding structure used in the above description of the invention: [0147] 1 GPC trace of batch P1 (Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester) [0148] 3 GPC trace of batch P2 (Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester) [0149] 5 GPC trace of batch P3 (Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester) [0150] 7 GPC trace of batch P4 (Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester) [0151] 9 GPC trace of batch P5 (Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-piperidine-4-carboxylic acid ethyl ester) [0152] 11 Storage modulus (G′) of batch P1 [0153] 13 Storage modulus (G′) of batch P2 [0154] 15 Storage modulus (G′) of batch P3 [0155] 17 Storage modulus (G′) of batch P4 [0156] 19 Storage modulus (G′) of batch P5 [0157] 21 Loss modulus (G″) of batch P1 [0158] 22 Loss modulus (G″) of batch P2 [0159] 23 Loss modulus (G″) of batch P3 [0160] 24 Loss modulus (G″) of batch P4 [0161] 25 Loss modulus (G″) of batch P5 [0162] 26 Loss modulus (G′) at 10° C. and 10 rad/s for batch P2 [0163] 27 Storage modulus (G′) at 10° C. and 10 rad/s for batch P2 [0164] 28 Storage modulus (G′) at 50° C. and 10 rad/s for batch P2 [0165] 29 Loss modulus (G′) at 50° C. and 10 rad/s for batch P2 [0166] 31 Flow curve for batch P2 [0167] 33 Storage modulus (G′) of batch P2 [0168] 34 Loss modulus (G″) of batch P2 [0169] 35 Dynamic viscosity of batch P2 (5 wt.-%) [0170] 37 Dynamic viscosity of batch P2 (10 wt.-%) [0171] 41 Dynamic viscosity of batch P2 (12.5 wt.-%) [0172] 43 Dynamic viscosity of batch P2 (15 wt.-%) [0173] 45 Dynamic viscosity of batch P2 (17.5 wt.-%) [0174] 47 Dynamic viscosity of batch P2 (20 wt.-%) [0175] 49 Dynamic viscosity of batch P2 (30 wt.-%) [0176] 51 comparison curve for F127 (10 wt.-%) [0177] 53 SANS Scattering data for batch P2 (6.9° C.) [0178] 55 SANS Scattering data for batch P2 (21.1° C.) [0179] 57 SANS Scattering data for batch P2 (30.0° C.) [0180] 59 SANS Scattering data for batch P2 (36.2° C.) [0181] 61 SANS Scattering data for batch P2 (39.7° C.) [0182] 63 correlation length for batch P2 [0183] 65 characteristic domain size d for batch P2 [0184] 67 bar chart dose-dependent cytotoxicity [0185] 69 bar control sample [0186] 71 bar blank sample [0187] 73 bar sample with 0.02 wt.-% [0188] 75 bar sample with 1 wt.-% [0189] 77 bar sample with 5 wt.-% [0190] 79 bar sample with 10 wt.-% [0191] 81 bar chart cell viability of NIH 3T3 fibroblasts [0192] 83 PI staining bars [0193] 85 bar control sample [0194] 87 bar methanol treated sample [0195] 89 bar of cells in 25% of gel [0196] 91 FDA staining bars [0197] 93 bar control sample [0198] 95 bar methanol treated sample [0199] 97 bar of cells in 25% of gel [0200] 99 curve control sample (for PI staining) [0201] 101 curve of cells in 25% of gel (for PI staining) [0202] 103 curve methanol treated sample (for PI staining) [0203] 105 curve control sample (for FDA staining) [0204] 107 curve methanol treated sample (for FDA staining) [0205] 109 curve of cells in 25% of gel (for FDA staining) [0206] 111 temperature dependence of storage modulus G′ for batch P2 [0207] 113 temperature dependence of loss modulus G′ for batch P2 [0208] 115 phase diagram of batch P2 [0209] 117 Storage modulus (G′) of batch P1a (Methyl-P[nPrOzi.sub.100-b-MeOx.sub.100]-piperidine-4-carboxylic acid ethyl ester) [0210] 119 Loss modulus (G″) of batch P1a [0211] 121 Storage modulus (G′) of batch P2a (Methyl-P[nPrOzi.sub.100-b-MeOx.sub.100]-tert-butyl-piperazine-1-carboxylat [0212] 123 Loss modulus (G′″) of batch P2a [0213] 125 Storage modulus (G′) of batch P3a (Propynyl-P[nPrOzi.sub.100-b-MeOx.sub.100]-piperidine-4-carboxylic acid ethyl ester) [0214] 127 Loss modulus (G′″) of batch P3a [0215] 129 Storage modulus (G′) of batch P4a (Propynyl-P[nPrOzi.sub.100-b-MeOx.sub.100]-methyl 3-mercaptopropionate) [0216] 131 Loss modulus (G′″) of batch P4a [0217] 133 Storage modulus (G′) of batch P5a (Propynyl-P[nPrOzi.sub.100-b-MeOx.sub.100]-hydroxy) [0218] 135 Loss modulus (G′″) of batch P5a [0219] 137 GPC trace of batch P1a [0220] 139 GPC trace of batch P2a [0221] 141 GPC trace of batch P3a [0222] 143 GPC trace of batch P4a [0223] 145 GPC trace of batch P5a [0224] 147 Storage modulus (G′) of batch P6a (modified Methyl-P[nPrOzi.sub.50-b-MeOx.sub.50]-tert-butyl-piperazine-1-carboxylat) [0225] 149 Loss modulus (G′) of batch P6a [0226] 151 Light microscope image of a printed constructs composed of orthogonal stacks of hydrogel strands [0227] 153 cell loaded constructs [0228] 157 results of FACS analysis on the influence of the printing process on the viability of NIH 3T3 fibroblasts [0229] 159 cells incorporated into the material and printed [0230] 161 cells incorporated into the material without further processing [0231] 163 untreated control cells
