Peptide capable of forming a gel for use in tissue engineering and bioprinting

Abstract

The present invention relates to peptides capable of forming a gel and to their use in tissue engineering and bioprinting. The present invention furthermore relates to a gel comprising a peptide in accordance with the present invention, to a method of preparing such gel and to the use of such gel. In one embodiment, such gel is a hydrogel. The present invention furthermore relates to a wound dressing or wound healing agent comprising a gel according to the present invention and to a surgical implant or stent comprising a peptide scaffold formed by a gel according to the present invention. Moreover, the present invention also relates to a pharmaceutical and/or cosmetic composition, to a biomedical device or an electronic device comprising the peptide according to the present invention.

Claims

1. A peptide capable of forming a gel by self-assembly, wherein the said peptide is selected from the group consisting of TABLE-US-00003 (SEQ ID NO: 9) IVFD, (SEQ ID NO: 10) IVOD, (SEQ ID NO: 17) IVFE, (SEQ ID NO: 18) IVOE, (SEQ ID NO: 25) IVFS, (SEQ ID NO: 26) IVOS (SEQ ID NO: 33) IVFR, (SEQ ID NO: 34) IVOR, (SEQ ID NO: 41) IVF(Dab), (SEQ ID NO: 42) IVO(Dab), (SEQ ID NO: 49) IVF(Dap), (SEQ ID NO: 50) IVO(Dap), (SEQ ID NO: 57) IVF(Orn), (SEQ ID NO: 58) IVO(Orn), (SEQ ID NO: 65) KFVI, (SEQ ID NO: 66) KOVI, (SEQ ID NO: 73) DFVI, (SEQ ID NO: 74) DOVI, (SEQ ID NO: 81) EFVI, (SEQ ID NO: 82) EOVI, (SEQ ID NO: 89) SFVI, (SEQ ID NO: 90) SOVI, (SEQ ID NO: 97) RFVI, (SEQ ID NO: 98) ROVI, (SEQ ID NO: 105) (Dab)FVI, (SEQ ID NO: 106) (Dab)OVI, (SEQ ID NO: 113) (Dap)FVI, (SEQ ID NO: 114) (Dap)OVI, (SEQ ID NO: 121) (Orn)FVI, and (SEQ ID NO: 122) (Orn)OVI, wherein I is isoleucine, L is leucine, V is valine, F is phenylalanine, K is lysine, D is aspartic acid, E is glutamic acid, S is serine, R is arginine, O is cyclohexylalanine, (Dab) is 2,4-diaminobutyric acid, (Dap) is 2,3-diaminopropionic acid, and (Orn) is omithine.

2. The peptide according to claim 1, being stable in aqueous solution at physiological conditions at ambient temperature for at least 6 months and/or being stable in aqueous solution at physiological conditions, at a temperature up to 90° C., for at least 1 hour.

3. A hydrogel or organogel comprising the peptide of claim 1.

4. The hydrogel or organogel according to claim 3, wherein: the hydrogel is stable in aqueous solution at ambient temperature for at least 1 month, the hydrogel is characterized by a storage modulus G′ to loss modulus G″ ratio that is greater than 2 to 5, the hydrogel or organogel is characterized by a storage modulus G′ from 500 Pa to 200,000 Pa at a frequency in the range of from 0.1 Hz to 100 Hz, and/or the hydrogel or organogel is characterized by a storage modulus G′ from 500 Pa to 200,000 Pa at a frequency in the range of from 0.1 Hz to 100 Hz.

5. The hydrogel or organogel according to claim 3, wherein the hydrogel or organogel has a higher mechanical strength than collagen or its hydrolyzed form (gelatin).

6. The hydrogel or organogel according to claim 3, wherein the hydrogel or organogel is comprised in at least one of a fuel cell, a solar cell, an electronic cell, a biosensing device, a medical device, an implant, a pharmaceutical composition and a cosmetic composition.

7. The hydrogel or organogel according to claim 3, which is injectable.

8. A method of preparing a hydrogel or organogel, the method comprising dissolving a peptide according to claim 1 in an aqueous solution or an organic solution, respectively.

9. The method of claim 8, wherein the dissolved peptide in aqueous or organic solution is further exposed to temperature, wherein the temperature is in the range from 20° C. to 90° C. and/or wherein the peptide is dissolved at a concentration from about 1 mg/ml to about 20 mg/ml.

10. A kit of parts, the kit comprising a first container with a peptide according to claim 1 and a second container with an aqueous or organic solution.

11. A peptide capable of forming a gel by self-assembly, said peptide having a sequence selected from the group consisting of: TABLE-US-00004 (SEQ ID NO: 1) IVFK, and  (SEQ ID NO: 2) IVOK, wherein I=isoleucine, V=valine, F=phenylalanine, K=lysine, and O=cyclohexylalanine.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) To further illustrate the technical features of embodiments of the present invention more clearly, the accompanying drawings provided for describing various embodiments are introduced briefly in the following. They are merely exemplary embodiments of the present invention and are not intended to limit it. Modifications on these embodiments are possible without departing from the scope of the present invention as defined in the claims.

(2) FIG. 1A shows ultrashort peptides IVZK and IVFK self-assemble into macromolecular nanofibrous hydrogels.

(3) FIG. 1B shows the chemical structures of the ultrashort peptides IVZK and IVFK and corresponding hydrogels as well as high resolution transmission electron microscopy (TEM) images.

(4) The average fiber diameter of IVFK peptide was calculated by plotting the distribution curve using the data collected from 13 TEM images. The average diameter is 10.3 nm (left low panel).

(5) The IVZK TEM shows three different types of average diameters (4.0 nm, 8.6 nm and 15.5 nm) (right low panel). The number of images used to calculate the average diameter were 10.

(6) The fibers diameter with 8.6 nm were the most abundant ones as compared to other fiber diameters. The single monomer fiber has an average diameter of 4.0 nm.

(7) FIG. 2 shows morphological characterization of the self-assembling peptide IVFK and IVZK hydrogels by FESEM showing different magnifications.

(8) FIG. 3 shows high mechanical strength was demonstrated for the peptide hydrogels. Storage moduli (G′) of different hydrogels (20 mg/mL) as a function of angular frequency under 0.1% strain, at 25° C. Frequency sweep was performed at 0.1% strain.

(9) FIG. 4 shows cell morphology of HeLa cells, HEK 293Tcells, and human dermal fibroblasts used for 3D cell culturing on peptide hydrogels.

(10) FIG. 5 shows graphical presentation of the MTT biocompatibility assay of HeLa with IVFK peptide.

(11) FIG. 6 shows Live/Dead staining of HeLa cells treated with IVFK peptide.

(12) FIG. 7 shows graphical presentation of the MTT biocompatibility assay of HeLa with IVZK peptide.

(13) FIG. 8 shows Live/dead images of HeLa cells treated with IVZK peptide.

(14) FIG. 9 shows graphical presentation of the MTT biocompatibility assay of HEK 293 T with IVFK peptide.

(15) FIG. 10 shows 3D Cell Viability Assay of Bioprinted Constructs. Live/Dead Staining of HEK 293 T cells treated with IVFK peptide

(16) FIG. 11 shows 3D Cell Viability Assay of Bioprinted Constructs. Live/Dead staining of HEK 293 T cells treated with IVFK peptide.

(17) FIG. 12 shows graphical presentation of the MTT biocompatibility assay of HEK 293 T with IVZK peptide.

(18) FIG. 13 shows 3D cell viability assay of bioprinted constructs. Live/Dead staining of HEK 293 T cells treated with IVZK peptide.

(19) FIG. 14 shows 3D cell viability assay of bioprinted constructs. Live/Dead staining of HEK 293 T cells treated with IVZK peptide.

(20) FIG. 15 shows peptide hydrogels cultured with human myoblast cells for one week.

(21) FIG. 16 shows 3D bioprinting of human dermal fibroblast cells using IVZK peptide solution as bioink (human dermal fibroblast cells—4 million/ml; peptide—10 mg/ml).

(22) FIG. 17 shows fluorescent images of human dermal fibroblasts embedded IVZK peptide hydrogels (human dermal fibroblast cells—4 million/ml; peptide—10 mg/ml). Actin cytoskeletal staining in green using phalloidin, nucleus staining in blue using DAPI.

(23) FIG. 18 shows fluorescent images of 3D human dermal fibroblasts embedded IVZK peptide hydrogels (human dermal fibroblast cells—4 million/ml; peptide—10 mg/ml). Actin cytoskeletal staining in green using phalloidin, nucleus staining in blue using DAPI.

(24) FIG. 19 shows fluorescence confocal microscopy images of 3D bioprinted human bone marrow-derived mesenchymal stem cells (BM-MSCs) cells using IVZK bioink at different days of cell culturing (nucleus is shown in blue, F-actin is shown in red and vinculin is shown in green).

(25) Furthermore, embodiments of the invention are now further described by reference to the following examples which are given to illustrate, not to limit the present invention.

EXAMPLES

(26) Peptide Synthesis

(27) Peptides were manually synthesized using solid phase peptide synthesis and purified to above 95% via HPLC. Amino acid and peptide content analysis were performed. Peptide molecules were synthesized manually using standard solid peptide synthesis on MBHA Rink Amide resin. DCM was used to swell the resin inside the reaction vessel for 30 min. Then, the solvent was removed by applying vacuum to the vessel. The Fmoc-protected group on the resin was removed by treating the resin with 10 mL of 20% (v/v) piperidine/DMF solution for 20 min. DMF and DCM were subsequently used to rinse the resin after each reaction to remove the excess materials from the vessel. Then, amino acid residue solution containing of 3 equivalents of N-protected amino acid, 2.9 equivalents of TBTU and 6 equivalents of DIPEA for each 1 equivalent mol of resin was poured and agitated for at least 2 h. Kaiser Test was performed after each coupling to examine the success of peptide coupling. The Fmoc protected group of the N-terminal peptide sequence was removed by adding 10 mL of 20% (v/v) piperidine/DMF and agitating for 20 min. These sequential steps that were amino acid coupling, washing, Kaiser Test, and Fmoc cleavage were repeated until the last sequence of amino acids. Acetylation was performed by pouring a mixed solution of acetic anhydride, DIPEA, and DMF to the vessel in order to cap the peptide sequence. The peptide sequence was then cleaved from the resin by mixing with acid solution that contained of 95% TFA, 2.5% water, and 2.5% triisopropylsilane for 2 h. The resin then was rinsed only with DCM and collected into the round bottom flask. The removal of excess TFA and DCM from the collected peptide solution was carried out by using rotary evaporation. Diethyl ether was added into the flask to disperse the peptide and kept for overnight. This solution then was centrifuged to separate the solid form of peptide from diethyl ether. The collected white solid was subsequently dissolved in water and freeze-dried to get the fluffy form of peptide. Finally, the peptide was purified by using prep-HPLC before being used.

(28) 9-Fluorenylmethoxycarbonyl (Fmoc) protected amino acids, Rink Amide MBHA resin, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) and 1-hydroxy-7-azabenzotriazole (HOAt) were purchased from GL Biochem. N,N-dimethylformamide (DMF), dichloromethane (DCM), diethyl ether, N,N-diisopropylethylamine (DIPEA), piperidine, and triisopropylsilane were purchased from Sigma-Aldrich. Acetic anhydride, dimethyl sulfoxide (DMSO), acetonitrile were purchased from Fisher Scientific. Trifluoroacetic acid (TFA) were purchased from Acros. The chemicals were used as received, without any purification.

(29) Peptide Hydrogel Preparation

(30) Lyophilized peptides were dissolved in milliQ water and vortexed to get a homogenous solution. Subsequently, 10× phosphate buffered saline at the final concentration of 1× was added to the peptide solution and vortexed shortly. Gelation occurred within few seconds to minutes or hours depending on the peptide sequence.

(31) Rheology Analysis

(32) The mechanical strength of the peptide hydrogels was measured using ARES-G2 rheometer (TA instruments) with an 8 mm parallel plate geometry at 22° C. 150 μL hydrogels were prepared inside a 9.6 mm diameter of polypropylene ring in which the top and bottom of each ring cast was covered with parafilm and kept inside a sealed tissue culture dish for overnight. After loading the hydrogel on peltier plate of the rheometer, water was dropped on the surrounded area to suppress the evaporation of the hydrogel. The gap measurement was adjusted between 1.6 and 1.9 mm. Time sweep measurement was performed prior to frequency sweep and amplitude sweep for 900 s duration with constant strain and angular frequency of 0.1% and 1 rad/s, respectively. The modulus values at the end of time sweep analysis were used as the standard equilibrium modulus for each hydrogel. Oscillatory frequency-sweep analysis were performed with 1% strain for a range of 0.1-100 rad/s. Oscillatory amplitude-sweep measurements were done with the constant angular frequency of 1 rad/s from 0.1 to 100% strain.

(33) Transmission Electron Microscopy (TEM) Studies

(34) The TEM studies were carried out on two different instruments; Tecnai G2 Spirit Twin with accelerating voltage of 120 kV and FEI Titan G2 80-300 CT with 300 kV emission gun. The Cryo TEM imaging was performed in low dose mode by using FEI's Titan Krios operating at 300 kV. The TEM samples for peptide nanofiber were prepared from diluted peptide hydrogel in water. One drop of this solution was then introduced on a carbon coated copper grid that has been treated with glow discharge plasma before being used. The drop was then kept for 10 minutes before being blotted using filter paper. To get a better contrast, the grid was stained using 2% uranyl acetate for 1 minute and then was dried for at least one day before imaging. The diameter of IVFK and IVZK peptide nanofibers were measured using an image-analysis software ImageJ from 13 and 10 TEM images, respectively. A size distribution histogram for each peptide nanofiber was created in Origin to calculate the average diameter of both peptides. See e.g. FIG. 1B.

(35) FESEM Analysis

(36) Peptide hydrogels were shock frozen in liquid nitrogen and immediately stored at −80° C. overnight. The samples were then vacuum dried in a freeze dryer (Labconco, USA) for 2-3 days. Subsequently, the lyophilized hydrogel samples were fixed onto an aluminium sample holder using conductive carbon tape and sputtered with platinum in a sputter coater. Three rounds of coating were performed at different angles to ensure complete coating. The surface of interest was then examined with a field-emission scanning electron microscope (FEI Nova Nano630 SEM, Oregon, USA) using an accelerating voltage of 5-10 kV or was visualized using FEI Quanta 200 FEG SEM with an accelerating voltage of 2-3 kV. The freeze-dried samples were adhered to carbon conductive tape on SEM stub and sputter coated with 3 nm of Platinum.

(37) Cell Culture

(38) Cells were cultured in medium 106 (Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The cells were maintained either in a T175 or T75 cell culture flask (Corning, USA) at 37° C. in a humidified incubator with 95% air and 5% CO2. The cells were subcultured by trypsination at approximately 80% confluence. The culture media was replenished every 48 hours.

(39) MTT Assay

(40) Biocompatibility studies were carried out in 96-well plates (Corning, USA). HDFn cells (10,000 cells/well) were seeded into a 96-well plate and incubated overnight in 200 μL complete growth medium. After incubation, media in the wells were changed. Peptides were weighed out and dissolved in milliQ water. To test the compatibility, peptides at different concentrations viz 5 mg/mL, 4 mg/mL, 2 mg/mL, 1 mg/mL and 0.5 mg/mL were added to the wells. Untreated wells were used as positive controls. The plates were incubated for 24 hours. Cell viability was determined by means of a colorimetric microculture assay (Vybrant® MTT Cell Proliferation Assay Kit, Thermo Fisher Scientific, USA) according to the manufacturer's protocol. Briefly, the plates were taken, the medium was carefully removed and fresh serum free medium containing 10% MTT reagent was added. After 2 h of incubation at 37° C., the supernatant media was removed and 200 μL of DMSO was added to each wells to dissolve the formazan crystals. Finally, the absorption of individual wells was read at 540 nm using a plate reader (PHERAstar FS, Germany).

(41) Live/Dead Staining

(42) Cells were seeded and treated with peptides according to the protocol described above. After 24 h of incubation, the spent media were removed and replaced with DPBS solution containing approximately 2 mM calcein AM and 4 mM ethidium homodimer-1 (LIVE/DEAD® Viability/Cytotoxicity Kit, Life Technologies™) and incubated for 40 min in dark. Before imaging, the staining solution was removed and fresh DPBS was added. Stained cells were imaged under an inverted confocal microscope (Zeiss LSM 710 Inverted Confocal Microscope, Germany).

(43) Cells (25,000 cells/plate) were embedded in 3D culture with hydrogel in a glass based confocal dish and incubated for 24 h using untreated cells as controls. After 24 h, the medium replaced with PBS (1×) solution containing approximately 2 μM calcein and 4 μM ethidium homodimer-1 (LIVE/DEAD Viability/Cytotoxicity Kit, Molecular Probe, L3224) and incubated for 30 min. Live cells were imaged in the green channel and dead cells in the red channel using ZEISS fluorescence microscope. The obtained pictures were superimposed using ImageJ as shown in FIG. 8.

(44) Cytoskeletal Staining

(45) Immunostaining was performed after 24 h of culture. In brief, the cells were fixed with 3.7% paraformaldehyde solution for 30 minutes and incubated in a cold cytoskeleton buffer (3 mM MgCl2, 300 mM sucrose and 0.5% Triton X-100 in PBS solution) for 10 minutes to permeabilise the cell membranes. The permeabilised cells were incubated in blocking buffer solution (5% FBS, 0.1% Tween-20, and 0.02% sodium azide in PBS) for 30 minutes at 37° C., followed by incubation in antivinculin (1:100) for 1 hour at 37° C. and subsequently with anti-mouse IgG (whole molecule)-FITC and rhodamine-phalloidin (1:200) for 1 hour at 37° C. Further, the cells were incubated in DAPI for 1 hour at 37° C. to counterstain the nucleus. These fluorescent dye treated cells were observed and imaged using laser scanning confocal microscope (Zeiss LSM 710 Inverted Confocal Microscope, Germany).

(46) 3D Culture of Cells in Peptide Hydrogels

(47) Cells were encapsulated in peptide hydrogels in 24 well tissue culture plates. Peptide solutions were added to the plate at 200 μL per well. Cells were resuspended in 3×PBS were added to each well at 100,000 cells/well and gently mixed. The final concentration of the peptide hydrogel was 1× after the addition of 3×PBS containing cells. Gelation occurred within 3-5 minutes and subsequently, culture medium was added to the wells. At pre-determined time points, the 3D cell viability assay, live/dead assay and cytoskeletal staining were performed.

(48) 3D cell culture was performed by making base on the bottom of plate to make sure that the cells grow in 3D construct, the base was made by pipetting peptide solution (peptide with water) into confocal plate and mixed with (PBS). After gel formation, peptide solution was added on top of this base. Cultured human dermal fibroblasts was trypsinzed, centrifuged and resuspended with PBS (2×) and mixed with peptide solution placed on top of the base. Once the gel form, DMEM was added on top as shown in FIG. 5 and incubate at 37° C. incubator and 5% CO2 for 48 hours and then different biocompatibility assays were applied on this construct.

(49) 3D Cell Proliferation Assay

(50) The CellTiter-Glo® luminescent 3D cell viability assay is a method to determine the number of viable cells in 3D hydrogels based on quantification of the ATP present, which signals the presence of metabolically active cells. After each time point, the hydrogels cultured with cells were washed twice with DPBS. Fresh medium was added to each well and equal amount of CellTiter-Glo® luminescent reagent was also added to the gels. The contents were mixed for 2 minutes to digest the hydrogels and then incubated for 10 minutes. After incubation, the luminescence was recorded using a plate reader (PHERAstar FS, Germany).

(51) 3D Bioprinting

(52) A 3D bioprinter was used to print constructs using peptide bioinks. To print peptide bioinks, the inventors designed a co-axial needle with three inlets. The top inlet was connected to 10×PBS solution, the right inlet was connected to cells suspended in serum free media and the left inlet was connected to peptide dissolved in milliQ water (15 mg/mL). The tubes, connectors and the co-axial nozzle were autoclaved before printing. While printing, three solutions were pumped into the nozzle with the aid of syringe pumps. The flow rate of peptide solution and cells solution were 25 μL/min while the flow rate of 10×PBS was maintained at 20 μL/min. Peptide solution mix with the cells and 10×PBS at the junction inside the co-axial nozzle just before the exit. Gelation occurs instantaneously and the peptide bioinks were printed. A simple ring structure was bioprinted in a layer-by-layer fashion with a diameter of about 8 mm and thickness about 2 mm. The constructs were printed onto 35-mm tissue culture petri dish. After bioprinting, the constructs were placed in biosafety cabinet for 3 min to further facilitate self-assembly of peptide bioinks. Then, the constructs were gently washed 2 or 3 times with culture medium. To each dish, 3 mL of culture medium was added and cultured in a humidified incubator at 37° C. and 5% CO2. At pre-determined time points, the constructs were taken out to perform 3D assay and cytoskeletal staining of cells as described above.

(53) The peptides according to the present invention are excellent gelators, and the gels formed can be used for many different purposes and applications. The features disclosed in the foregoing description, in the claims or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

(54) Further modifications of the preferred embodiments are possible without leaving the scope of the invention which is solely defined by the claims.