3D Printing and Drug Delivery

Abstract

A 3D structure of a hydrogel for supporting cell growth or for use in sustained drug delivery is formed using peptides and/or peptide derivatives that self-assemble via cross-linking into a stiff gel. The hydrogel structure is formed using a method based on 3D printing. A hydrogel precursor is extruded under conditions to generate a hydrogel, by extruding a solution of the peptides into a solution containing cations, whereby the cations enable cross-linking of pi-stacked peptides, or by co-extruding it with the cations, the peptides and cations being mixed only at the point of co-extrusion. The stiffness of the hydrogel can be tuned by adjusting the combination of peptides, either by the selection of peptides or combinations thereof, or the proportions of the combinations, and/or by adjusting the proportion of cations present.

Claims

1. A method of printing, comprising: a. preparing ink comprising a mixture of hydrogel precursor and activator in non-gelled form, b. printing the ink onto a substrate, and c. allowing the ink to gel, wherein the hydrogel precursor comprises a plurality of peptide derivatives and the activator comprises a cross-linking agent

2. A method according to claim 1, wherein preparing the ink comprises combining the hydrogel precursor with the activator so that it begins to gel, and printing the gelling ink before it has gelled.

3. A method according to claim 1, wherein preparing the ink comprises combining the hydrogel precursor with the activator so that it partially gels, and allowing the ink to gel comprises combining the partially gelled ink with further activator so that a gel is formed.

4. A method according to any preceding claim, wherein the ink has a viscosity of at least 150 cP.

5. A method according to any preceding claim, wherein the ink has a viscosity of at least 250 cP.

6. A method according to any preceding claim, comprising co-printing or coextruding hydrogel precursor and activator from separate reservoirs.

7. A method of printing, comprising a. preparing ink comprising a hydrogel precursor in non-gelled form, b. printing the ink so as to contact it with activator, and c. allowing the ink to gel, wherein the hydrogel precursor comprises a plurality of peptide derivatives and the activator comprises a cross-linking agent.

8. A method according to claim 7, comprising printing into a solution containing the activator.

9. A method according to claim 7, comprising co-printing or coextruding hydrogel precursor and activator from separate reservoirs.

10. A method according to any previous claim, wherein the peptide derivatives comprise at least 2 peptides linked to a third component which is an aromatic peptide or is an aromatic stacking ligand.

11. A method according to any previous claim, wherein the activator comprises a cross-linking agent, e.g. a cation.

12. A method according to any previous claim, wherein the peptide derivatives are of formula I
ASL-GA-GA-X.sub.(n) (I) wherein ASL is an aromatic stacking ligand comprising an aromatic group, each GA is independently an amino acid or a derivative thereof, X is an amino acid or a derivative thereof, n is an integer from 0 to 3, and wherein the peptide derivatives form a gel in the presence of crosslinking cations.

13. A method according to claim 12, wherein ASL is Fmoc, CBz, an aromatic amino acid, or a derivative thereof.

14. A method according to claim 12 or 13, wherein the peptides derivatives are of formula Ia
ASL-GA1-GA2-X.sub.(n) (Ia) wherein GA1 is selected from phenylalanine (F), tyrosine (Y) and tryptophan (W) and derivatives thereof, Each GA2 and X is independently selected from (1) neutral amino acids alanine (A), leucine (L), asparagine (N), methionine (M), cysteine (C), glutamine (Q), proline (P), glycine (G), serine (S), isoleucine (I), threonine (T), tyrosine (Y), tryptophan (W) and valine (V), positively charged amino acids arginine (R), histidine (H) and lysine (K), and negatively charged amino acids aspartic acid (D) and glutamic acid (E), and derivatives thereof, and (2) phenylalanine (F), tyrosine (Y) and tryptophan (W) and derivatives thereof, and n is an integer from 0 to 3.

15. A method according to claim 14, wherein the peptides comprise a mixture of peptides, some of which comprise a GA2 from list (1) and some of which comprise a GA2 from list (2).

16. A method according to any previous claim wherein the ink further comprises cells.

17. A method according to any previous claim, wherein the ink further comprises an active agent.

18. A method according to any previous claim, comprising printing a structure containing an active agent, for sustained delivery of the active agent.

19. A method according to any previous claim, comprising printing a cell support structure containing cells.

20. A method according to any previous claim, comprising printing a sheet of hydrogel precursor.

21. A method according to any previous claim, comprising printing a first layer of ink, allowing the ink to partially gel, and printing a second layer of ink in contact with the first layer.

22. A method according to any previous claim, wherein the gelled hydrogel has a stiffness of 5 kPa or greater.

23. A method according to any previous claim, wherein the gelled hydrogel has a stiffness of 10 kPa or greater.

24. Printer ink, comprising hydrogel precursor, wherein the hydrogel precursor comprises a plurality of peptide derivatives and forms a gel in contact with a cross-linking agent.

25. Printer ink according to claim 24, further comprising cells.

26. Printer ink according to claim 24 or 25, further comprising an active agent.

27. Printer ink according to any of claims 24 to 26, further comprising a cross-linking agent.

28. Printer ink according to any of claims 24 to 27, wherein the peptide derivatives comprise at least 2 peptides linked to a third component which is an aromatic peptide or is an aromatic stacking ligand.

29. A composition comprising hydrogel precursor and active agent for use in sustained release delivery of the active agent, wherein the hydrogel precursor comprises a plurality of peptide derivatives and forms a gel in contact with a cross-linking agent.

30. A composition for use according to claim 29, wherein the peptide derivatives each independently comprise at least 2 peptides linked to a third component which is an aromatic peptide or is an aromatic stacking ligand.

31. A composition for use according to claim 29 or 30, having a stiffness of 5 kPa or greater.

32. A composition for use according to claim 29 or 30, having a stiffness of 10 kPa or greater.

33. A composition for use according to any of claims 29 to 32, wherein the sustained release of the active agent is over a period of 2 days or more.

34. A composition for use according to any of claims 29 to 33, wherein the sustained release of the active agent is over a period of 5 days or more.

35. A composition for use according to any of claims 29 to 34 by injection of hydrogel precursor into a patient wherein the hydrogel gels in situ.

35. A composition for use according to any of claims 29 to 34 by surgical insertion of gelled hydrogel into a patient.

36. A method of sustained delivery of an active agent to a patient, comprising administering to the patient a hydrogel or precursor containing the active agent, wherein the hydrogel or precursor comprises a plurality of peptide derivatives and forms a gel in contact with a cross-linking agent.

37. A method according to claim 36, wherein the peptide derivatives each independently comprise at least 2 peptides linked to a third component which is an aromatic peptide or is an aromatic stacking ligand.

38. A method according to claim 36 or 37, wherein the hydrogel has a stiffness of 5 kPa or greater.

39. A method according to claim 38, wherein the hydrogel a stiffness of 10 kPa or greater.

40. A method according to any of claims 36 to 39, wherein the sustained release of the active agent is over a period of 2 days or more.

41. A method according to any of claims 36 to 40, wherein the sustained release of the active agent is over a period of 5 days or more.

42. A method according to any of claims 36 to 41, wherein administering is by injection of hydrogel precursor into a patient wherein the hydrogel gels in situ.

43. A method according to any of claims 36 to 41, wherein administering is by surgical insertion of gelled hydrogel into a patient.

44. A composition for use according to any of claims 29 to 35 or a method according to any of claims 36 to 40, wherein the peptides are of formula I
ASL-GA-GA-X.sub.(n) (I) wherein ASL is an aromatic stacking ligand comprising an aromatic group, each GA is independently an amino acid or a derivative thereof, X is an amino acid or a derivative thereof, n is an integer from 0 to 3, and wherein the peptide derivatives form a gel in the presence of crosslinking cations.

45. A composition for use according to claim 44 or a method according to claim 44, wherein ASL is Fmoc, CBz, an aromatic amino acid, or a derivative thereof.

46. A composition for use according to claim 44 or 45 or a method according to claim 44 or 45, wherein the peptides are of formula Ia
ASL-GA1-GA2-X.sub.(n) (Ia) wherein GA1 is selected from phenylalanine (F), tyrosine (Y) and tryptophan (W) and derivatives thereof, Each GA2 and X is independently selected from (1) neutral amino acids alanine (A), leucine (L), asparagine (N), methionine (M), cysteine (C), glutamine (Q), proline (P), glycine (G), serine (S), isoleucine (I), threonine (T), tyrosine (Y), tryptophan (W) and valine (V), positively charged amino acids arginine (R), histidine (H) and lysine (K), and negatively charged amino acids aspartic acid (D) and glutamic acid (E), and derivatives thereof, and (2) phenylalanine (F), tyrosine (Y) and tryptophan (W) and derivatives thereof, and n is an integer from 0 to 3.

47. A composition for use according to claim 46 or a method according to claim 46, wherein the peptides comprise a mixture of peptides, some of which comprise a GA2 from list (1) and some of which comprise a GA2 from list (2).

48. Apparatus for creating a cell growth support structure, comprising a container of printer ink according to any of claims 24 to 28, linked to an extruder capable of extruding the ink.

49. Apparatus according to claim 48, further comprising a container of activator, linked to an extruder capable of extruding the agent so that is contacts extruded precursor.

Description

SPECIFIC DESCRIPTION

[0166] To help understanding of the invention, specific embodiments thereof will now be described by way of example and with reference to the accompanying drawings in which:

[0167] FIG. 1 shows a double barrel syringe loaded with hydrogel precursor and calcium chloride solution for simultaneous dispersion;

[0168] FIG. 2 shows photographs of a gel formed from Run 5;

[0169] FIG. 3 shows a photograph of a three-way connector with two syringes attached;

[0170] FIG. 4 is a graph showing the viscosity of pre-gels prepared from lyophilised powder;

[0171] FIG. 5 is a graph showing the viscosity of pre-gels prepared from lyophilised powder and calcium chloride solution;

[0172] FIG. 6 shows the hydrogel from Run 10;

[0173] FIG. 7 is a graph showing stiffness differences (kPa) between the three gel types (soft, soft-to-firm and firm) and the stability over a short period of time;

[0174] FIG. 8 is a graph showing stiffness differences (kPa) between the three gel types (soft, soft-to-firm and firm) and the stability over a short period of time;

[0175] FIG. 9 is a graph showing the viscosity results of potential bio-inks prepared from different combinations of materials;

[0176] FIG. 10 shows a photograph of printing using Cellink Inkredible printer with an ink according to the invention;

[0177] FIG. 11 shows a photograph of printed gel grid structure (5 layers) from Run 14;

[0178] FIG. 12 shows a photograph of printed gel cylinder structure from Run 15;

[0179] FIG. 13 shows a photograph of printed gel grid structure (5 layers) from Run 15;

[0180] FIG. 14 is a graph showing viscometry results of bio-inks according to the invention pre and post freeze drying;

[0181] FIG. 15 is a photograph of a structure printed by the printer of FIG. 10 immediately after printing;

[0182] FIG. 16 is a graph of the release profile of Propranolol from 10 mM Fmoc-FF/S;

[0183] FIG. 17 is a graph of the cumulative release profile of Propranolol from 10 mM Fmoc-FF/S;

[0184] FIG. 18 is a graph of the release profile of Betaxolol from 10 mM Fmoc-FF/S;

[0185] FIG. 19 is a graph of the cumulative release profile of Betaxolol from 10 mM Fmoc-FF/S;

[0186] FIG. 20 is a graph of the release profile of Quinidine from 10 mM Fmoc-FF/S; and

[0187] FIG. 21 is a graph of the cumulative release profile of Quinidine from 10 mM Fmoc-FF/S.

EXAMPLE 1

[0188] Two methods of printing using hydrogels were investigated. The first used pre-gel mixed with CaCl.sub.2 solution to produce a partially crosslinked material which was dispensed into a concentrated CaCl.sub.2 solution to rapidly fully gel the material in a 3D structure. The second comprised dispensing pre-gel with CaCl.sub.2 solution via a double barrel syringe to immediately cause cross-linking and for a 3D structure to be created.

[0189] Preparation of the Solutions.

[0190] Hydrogel Precursor Solution

[0191] Fmoc-FF/S (i.e. a mixture of Fmoc-FF and Fmoc-S) lyophilised powder (batch produced using 91% pure Fmoc-FF for investigation purpose only) was weighed into a 50 mL tube, which had been tared on the balance. To obtain hydrogel precursors with concentrations of 10, 20 and 30 mM, 0.22, 0.44 and 0.66 grams were used and reconstituted in sterile water. Thorough mixing and sonication for 30 seconds was performed using the vortex and sonicator water bath. Pre-gels were stored at 4 C. until further use.

[0192] Calcium Chloride Solution

[0193] Calcium Chloride solution was prepared at 5, 20 and 100 mM concentrations by weighing out 0.055, 0.222 and 1.110 grams of calcium chloride into a beaker and making the volume up to 100 mL with sterile water. A magnetic bead and stirring platform was used to dissolve the calcium chloride by mixing for 10 minutes. The solutions were then 0.2 m syringe filtered into a clean glass beaker and stored at 4 C. until further use.

[0194] 3D Structure Formation

[0195] The 3D structures were formed using the double barrel syringe shown in FIG. 1. The rubber pistons supplied with the syringe, were attached to the plunger with adhesive so that they could be used multiple times. Hydrogel precursor was loaded into one side of the syringe and a CaCl.sub.2 solution in the other side. The plunger with pistons attached was inserted and the syringe cap was removed so that a 200 L tip could be placed on.

[0196] The mix of pre-gel/CaCl.sub.2 solution used was as detailed in the following table;

TABLE-US-00001 TABLE 1 Pre-Gel Concentration of Run Concentration (mM) CaCl.sub.2 (mM) 1 10 20 2 10 100 3 20 20 4 20 100 5 30 20 6 30 100

[0197] Results:

[0198] The original experiments used pre-gel and CaCl.sub.2 solutions incubated at 37 C., 5% CO.sub.2 prior to use and returned to these conditions between use. The glass container with 3D structure was incubated at the same conditions (37 C., 5 % CO.sub.2). Subsequently, the work was performed at room temperature, to demonstrate that 3D structure formation is not temperature dependent.

[0199] A cube was used to demonstrate 3D structure capabilities of the peptide gel. A section of paper was placed under the glass container with a square (2 cm.sup.2) drawn on it and was used as a template for the 3D cube. The 3D structure was created by moving the dispensing syringe, in effect the nozzle, from top left to bottom, across then up to top right corner of the square template into the glass container. This was repeated three times in a continuous flow so as the same volume of material was dispensed for each pre-gel and CaCl.sub.2 solution combination.

[0200] Structures

[0201] Run 1, namely 10 mM pre-gel+20 mM CaCl.sub.2 solution, resulted in a cube although some of the material was not attached to the main body as the gel took a few seconds (2-3 s) to form a solid gel.

[0202] Run 2, namely 10 mM pre-gel+100 mM CaCl.sub.2 solution, resulted in a cube although some of the material was again detached from the main body as the gel look a few seconds (2-3 s) to form as a solid gel. It was also observed that the stronger CaCl.sub.2 solution resulted in a more opaque gel material.

[0203] Runs 4 and 3, namely 20 mM pre-gel+100 mM CaCl.sub.2 solution and 20 mM pre-gel +20 mM CaCl.sub.2 solution respectively resulted in similar results as for the 10 mM pre-gel material but with the gel material forming slightly quicker (1-2 s).

[0204] Run 5, as shown in FIG. 5, namely 30 mM pre-gel+20 mM CaCl.sub.2 solution, resulted in a cube with a lot less of the material not attached to the main body as the gel. The gel material formed very quickly and seemed to gel almost instantaneously to form a solid 3D cube.

[0205] Run 6, namely 30 mM pre-gel+100 mM CaCl.sub.2 solution, resulted in a cube again with a lot less of the material not attached to the main body as the gel. The gel material formed very quickly and seemed to gel almost instantaneously to form a solid 3D cube. The stronger CaCl.sub.2 solution resulted in a more opaque gel material.

[0206] Conclusion:

[0207] The first method of pre-setting the gel was used initially then refined as the second method (see discussion below). Note that some methods utilised also encapsulated individual cells in small volumes of gel.

[0208] The second method was successful for producing a 3D cube structure built up from many extruded layers, gelled on top of each other. With more controlled dispensing systems more defined and more complex structures were achievable. The 3D cubes made retained the cube shape and when immersed in water again retained their shape.

[0209] The invention hence provides a method of creating 3D hydrogel structures using a technique akin to 3D printing.

EXAMPLE 2

[0210] Following on from Example 1, a second experimental procedure was devised and used to dispense pre-gel and CaCl.sub.2 solution from two separate syringes through a 3-way connector (see FIG. 3). The two components were dispensed through the third opening on the connector via a 1 mL pipette tip, which had been attached. The gel material produced was tested for rheology over a 5 hour period to demonstrate short term stability.

[0211] Having identified the concentration of calcium chloride solution required to initiate quick gelation of the peptide derivatives, initial investigation into the mechanical properties of the structures formed under these new conditions was carried out, and an assessment of the tunability of the new bio-ink.

[0212] With regards to specific mechanical properties that were studied, initially the viscosity of the bio-ink was investigated as this is a key component in determining its compatibility with 3D bioprinting techniques.

[0213] The stiffness of the fully cross-linked printed constructs were also measured using rheology. This is an important characteristic, as not only will it have an effect on the stability of the printed structure, as successive layers of material are deposited, but the stiffness will also have an influence over the behaviour of cells incorporated into the gel, with tunability being a highly attractive feature within 3D cell culture. As gelation of these new bio-ink gels have been triggered under alternative conditions to standard cell culture protocols, the stiffness of the gels were found to be different.

[0214] Preparation of the Solutions

[0215] Fmoc-FF/S lyophilised powder (batch produced using 91% pure Fmoc-FF for investigation purpose only) was weighed into a 50 mL tube. To obtain pre-gels with concentrations of 10, 20, 30, 50, 80, 100, 200 and 300 mM, 0.22, 0.44, 0.66, 1.1, 1.76, 22, 44 and 66 grams were used and reconstituted in sterile water. Thorough mixing and sonication for 30 seconds was performed using the vortex and sonicator water bath. Pre-gels were stored at 4 C. until further use. Calcium chloride solutions were prepared at 0.5, 2, 5 and 10 mM concentrations as it was believed these concentrations would achieve partial crosslinking but not full gelation.

[0216] The viscosity results of pre-gel alone and pre-gel mixed with calcium chloride solution are detailed in the following tables;

TABLE-US-00002 TABLE 2 Viscosity Centipoise Pre-Gel Concentration (mM) R1 R2 R3 Average 10 5.0 4.2 3.5 4.2 20 6.8 7.2 6.8 6.9 30 10.8 9.9 9.5 10.1 50 113.1 910.0 906.0 98.2 80 116.6 111.0 140.0 122.5 100 134.0 131.0 146.0 137.0 200 213.2 222.0 239.0 224.8 300 238.7 296.0 306.0 280.1

TABLE-US-00003 TABLE 3 Pre-Gel Concentration (mM) + Viscosity Centipoise CaCl.sub.2 Solution (mM) R1 R2 R3 Average 20 + 2 14.2 12.7 12.1 13.0 30 + 0.5 18.3 15.9 10.3 14.8 30 + 2 17.6 17.3 16.7 17.2 100 + 0.5 136.3 134.0 150.0 140.3 100 + 5 179.6 174.0 202.0 185.3 200 + 0.5 253.3 252.5 263.6 256.5 200 + 10 276.3 294.1 305.7 292.0

[0217] The graphs of FIG. 4 and FIG. 5 have been prepared from this data. An increase in pre-gel concentration in turn increased the viscosity of the material. The viscosity results also demonstrated that with addition of calcium chloride solution the material becomes more viscous compared to the same concentration of pre-gel with no calcium chloride solution addition.

[0218] Using this knowledge further 3D printing experiments were conducted using the pre-gel and CaCl.sub.2 solutions dispensed from two separate syringes through a three way connector as set out above.

[0219] Preparation of the Solutions

[0220] Pre-Gel Formation

[0221] Fmoc-FF/S lyophilised powder (batch produced using 91% pure Fmoc-FF for investigation purpose only) was weighed into a 50 mL tube. To obtain pre-gels with concentrations of 10, 20 and 30 mM, 0.22, 0.44 and 0.66 grams were used and reconstituted in sterile water. Thorough mixing and sonication for 30 seconds was performed using the vortex and sonicator water bath. Pre-gels were stored at 4 C. until further use.

[0222] Calcium Chloride Solution

[0223] Calcium Chloride solution was prepared at 20 and 100 mM concentrations by weighing out 0.222 and 1.110 grams of calcium chloride into a beaker and making the volume up to 100 mL with sterile water. A magnetic bead and stirring platform was used to dissolve the calcium chloride by mixing for 10 minutes. The solutions were then 0.2 m syringe filtered into a clean glass beaker and stored at 4 C. until further use.

[0224] 3D Structure Formation

[0225] Pre-gel mixed with a CaCl.sub.2 solution was found to immediately form crosslinked material.

[0226] As shown in FIG. 3, a 3-way syringe connector with two syringes attached, was loaded with pre-gel in one syringe and CaCl.sub.2 solution in the other. The plunger of the syringes were pressured at the same time so the mix of pre-gel and CaCl.sub.2 solution was a 50:50 ratio.

[0227] The mix of pre-gel/CaCl.sub.2 solution used was as detailed in the following Table 4:

TABLE-US-00004 TABLE 4 Pre-Gel Concentration of CaCl.sub.2 to Concentration obtain Fully Cross-linked 3D Run (mM) Structure (mM) 7 10 20 8 10 100 9 20 20 10 20 100 11 30 20 12 30 100

[0228] Appearance

[0229] A section of paper was placed under a glass container with a square (4 cm.sup.2) drawn on it and was used as a template for the 3D cube. The 3D structure was created by moving the 4-way connector with the two syringes from top left to bottom, across then up to top right corner of the square template into the glass container. This was repeated twice in a continuous flow so as the same volume of material was dispensed for each pre-gel and CaCl.sub.2 solution combination. Three different gel concentrations were selected 30 mM, 20 mM and 10 mM (representing firm, soft-to-firm and soft gels).

[0230] Images of successful 3D structures were captured immediately after their formation. The hydrogel formed in Run 7, produced a soft gel; the hydrogel in Run 8 produced a soft-to-firm gel; the hydrogel from Run 9 produced a firm gel; the hydrogel from Run 10 produced a soft gel (as shown in FIG. 6); the hydrogel from Run 11 produced a soft-to-firm gel; and the hydrogel from Run 12 produced a firm gel.

[0231] Rheology

[0232] Sections of the formed 3D cube were removed and rheology analysis was performed immediately after dispensing and at 30, 60, 90, 120, 180, 240 and 300 minutes.

[0233] FIG. 7 is a graph showing stiffness differences (kPa) between the three gel types (soft, soft-to-firm and firm), at the higher CaCl.sub.2 concentration, and the stability over a short period of time.

[0234] Frequency sweeps were carried out from 0.1 Hz to 100 Hz, at constant oscillation amplitude and temperature, and with a working gap of 0.5 mm. From the graph of storage modulus against frequency produced, the stiffness of the sample was calculated. The geometry of the circular reader was PU20 (20 mm diameter).

[0235] FIG. 8 is a graph showing stiffness differences (kPa) between the three gel types (soft, soft-to-firm and firm) at the lower CaCl.sub.2 concentration and the stability over a short period of time

[0236] Conclusion:

[0237] The described method used which simulates a two separate ink cartridge set-up for printing was successful in producing a 3D cube structure. The gel material formed more or less instantaneously and rheology analysis was performed on sections of the 3D cube over a five-hour period. The 3D cubes for all concentrations of pre-gel and calcium solution retained their shape and rheology results showed that stiffness of the gel was satisfactory and stable over the period of time it was monitored.

[0238] At the lower concentrations of calcium chloride (i.e. 0.5 and 2 mM) there was no impact on the appearance of the pre-gel. However, as the concentration of calcium chloride was increased this resulted in the pre-gel becoming more opaque. An increase in pre-gel concentration lead to an increase in viscosity, due to the increased density of fibres present. The addition of the calcium chloride also resulted in an increase in the viscosity due to the cross-linking (or partial cross-linking) of the fibres. The viscosity is also affected by the printing process, for example by the printer head nozzle size, the distance to printing surface and the temperature. As a result the exact concentration is determined empirically for an individual printer.

[0239] Building on this, development was continued, moving to the exploration of layered 3D printing to create tubular and lattice constructs, allowing further optimisation of both gel crosslinking strength and printing parameter control.

EXAMPLE 3

[0240] A third experimental procedure was then developed to use a 3D printer, and specifically one in which bio-ink is printed from a single nozzle from a single cartridge. Most 3D printers used in laboratories at the present time are fitted with a single print cartridge only; printers having two cartridges and thus able to print two materials simultaneously are more expensivealthough this may change as this technique develops. Above experiments demonstrate that this new bio-ink can be used in printers having two cartridges. This set of experiments also demonstrates that the new bio-ink can be used with printing having a single cartridge only.

[0241] The printer used was a Cellink Ink-credible 3D printer.

[0242] This was used to investigate layered 3D printing to create tubular and lattice constructs, and gel formulations including additional elements.

[0243] Aim

[0244] To produce a material that could be printed from a single printer cartridgethrough one nozzlewith no additional activator agent required at the point of printing.

[0245] This method was also used to investigated further combinations of bio-ink materials for their viscosity properties in comparison to two common printable materials, namely 4% alginate with 0.15% CaCl.sub.2, and 40% Pluronic F127. The specific combinations of bio-ink materials were tested for their ability to print specific 3D shapes.

[0246] The additional materials included in the different combinations were the laminin sequence IKVAV lyophilised powder; 4% alginate with 0.15% CaCl.sub.2; 40% Pluronic F127; DMEM media; PBS and Kolliphor P407.

[0247] Preparation of the Solutions

[0248] All samples were prepared in 1.5 mL Eppendorf tubes (with the exception of a scale-up sample which was prepared in a 15 mL falcon tube).

[0249] The viscosity when printed of various bio-inks was tested as set out in the table of FIG. 9. The constituents of the gets are set out in the Table and were all made as described below in relation to three examples.

[0250] Example 1-30 mM FFS with 0.25% (w/v) IKVAV addition+200 uL DMEM per 1 mL of pre-gel

[0251] Fmoc-FF and Fmoc-S were weighed so as to be present at a 1:1 molar ratio. 12.5 mg of IKVAV was added. The powders were dissolved and lyophilised.

[0252] 157 mg of Biogelx lyophilised FFS/IKVAV powder (as described above) was weighed into a 15 mL Falcon tube and 5 mL dH.sub.2O added. The tube was then vortexed and sonicated until no particulates remained. Following this, 1 mL of DMEM was added and the tube was vortexed again to mix. A final sonication was used to remove any pockets of air. The bio-ink was then be transferred to the printer cartridge.

[0253] Example 2-30 mM FFS in 1% Kolliphor P407+200 uL DMEM per 1 mL of pre-gel

[0254] A 20% stock solution of Kolliphor P407 was prepared by dissolving the powder in water at room temperature.

[0255] 132 mg of Fmoc-FF and Fmoc-S (1:1) lyophilised powder was weighed into a 15 mL Falcon tube and 5 mL dH.sub.2O added. The tube was then vortexed and sonicated until no particulates remain. Following this, 250 uL of the stock 20% Kolliphor P407 was added and the tube was vortexed to mix. 1 mL of DMEM was added and the tube was vortexed again. A final sonication was used to remove any pockets of air. The bio-ink could then be transferred to the printer cartridge.

[0256] Example 3-30 mM FFS+200 uL DMEM per 1 mL of pre-gel 132 mg of Fmoc-FF and Fmoc-S (1:1 lyophilised powder) was weighed into a 15 mL Falcon tube and 5 mL dH.sub.2O added. The tube was then vortexed and sonicated until no particulates remain. Following this, 1 mL of DMEM was added and the tube was vortexed again to mix. A final sonication was used to remove any pockets of air. The bio-ink could then be transferred to the printer cartridge.

[0257] Results

[0258] As shown in FIG. 9, the prior art materials which have the highest viscosities immediately after printing are the currently two commonly used printing materials, namely 4% alginate with 0.15% CaCl.sub.2 (which has a viscosity of 1583 Centipoise) and 40% Pluronic F127 (which has a viscosity of 4161 Centipoise). This allows them to hold their shape once printed.

[0259] For peptide-based gels of the invention, printable inks were obtained that were partially gelled and had initial viscosities of about 250-300 Centipoise or greater.

[0260] The resulting shortlisted library of combination materials were used to further explore their ability to create tubular and grid constructs using layered 3D printing by a 3D extrusion printer (CellinkInkredible). FIG. 10 shows a photograph of printing using Cellink Inkredible printer.

[0261] Printing fully gelled material resulted in the printer head acting as a filter, separating the water content of the gel from the fibres structure.

[0262] Printing the pre-gel on its own directly into a bath of DMEM media or printing pre-gel that had been reconstituted with diluted DMEM were both successful methods. For example a disturbed ring structure was obtained, and the material did gel. Thus, we used between 0 and 50% DMEM in subsequent experiments.

[0263] Following the further viscosity studies of combination materials it was decided that three specific combinations resulted in viscosities for further printing studies. These were 30 mM FFSIKVAV (namely Fmoc-FF, Fmoc-S and IKVAV) with 200 L DMEM added per 1 mL of pre-gel (Run 13 and as described in Example 1); 30 mM FFS (namely Fmoc-FF with Fmoc-S) in 1% Kolliphor P407 and 200 L DMEM added per 1 mL of pre-gel gel (Run 14 and as described in Example 2); and lastly 30 mM FFS and 200 L DMEM added per 1 mL of pre-gel gel (Run 15 and as described in Example 3).

[0264] These materials were made by rehydrating FFS powder (1:1 Fmoc-FF and Fmoc-S) in dH.sub.2O and then adding DMEM at 200 L per mL of pre-gel. For Kolliphor P407 addition a stock 20% solution had been prepared previously and this was diluted (e.g. 1 in 20) with the pre-gel to achieve different working concentrations (i.e. 1 in 20 dilution would give a 1% solution). Then DMEM addition would follow at the same volume as previously stated for the other two samples. All samples were left overnight at room temperature for these investigations.

[0265] Printed rings, grids and cylinders achieved for all samples by successively building up layers of material. FIG. 11 shows a photograph of printed gel grid structure (5 layers) from Run 14; FIG. 12 shows a photograph of printed gel cylinder structure from Run 15; and FIG. 13 shows a photograph of printed gel grid structure (5 layers) from Run 15. The pressure required to print all materials was 4-fold less than the material reconstituted with 50% diluted media which is preferred for when printing cells as high pressure is likely to damage them. The constructs were reviewed following 1 hour incubation at room temperature post printing, demonstrating that the structures held their shape without additional cross-linker.

[0266] Peptide derivatives are stable as lyophilised powders and the provision of bio-inks in the form of powders, which can be made up by the addition of water, or culture media, represent a stable and convenient form for end users. As a result bio-inks that have the potential to be prepared for storage and transport in the form of a powder were investigated.

[0267] Bio-inks were created based on the materials discussed above, and two further combinations, namely one containing RGD (i.e. the tripeptide arginine-glycine-aspartic acid) and the other GrowDex (i.e. hydrogel extracted from birch, and available from www.growdex.com). Bio-inks were created containing different combinations of components and were freeze dried into a lyophilised powder.

[0268] The following bio-inks were prepared:

[0269] A 30 mM FFS [0270] 52.8 mg of lyophilised FFS powder (FFS017MC) (ie a 1:1 lyophilised mixture of Fmoc-FF and Fmoc-S) was weighed into a tared 7 mL glass vial and 2 mL dH.sub.2O was pipetted into the vial. Vortex and sonication was used to dissolve/rehydrate the powder. 400 L DMEM was added by pipette to the vial and mixed by vortex, sonication was used to remove air bubbles.

[0271] B 30 mM FFSIKVAV (0.25% w/v) [0272] 63.2 mg of lyophilised FFS and IKVAV powder (FFSIKVAV001 MC) (i.e. a lyophilised mixture of 1:1 Fmoc-FF and Fmoc-S with 0.25% IKVAV) was weighed into a tared 7 mL glass vial and 2 mL dH.sub.2O was pipetted into the vial. A magnetic bead was placed in the vial and the vial was placed on to a stirring platform which was used to dissolve/rehydrate the powder. 400 L DMEM was added by pipette to the vial and it was mixed again using the magnetic bead, sonication was used to remove air bubbles.

[0273] C 30 mM FFSRGD (ratio 2:1:1) [0274] 86.9 mg of lyophilised FFS and RGD powder (FFSRGD001MC) was weighed into a tared 7 mL glass vial and 2 mL dH.sub.2O was pipetted into the vial. Vortex and sonication was used to dissolve/rehydrate the powder. 400 L DMEM was added by pipette to the vial and mixed by vortex, sonication was used to remove air bubbles.

[0275] D 30 mM FFS in 0.83% Kolliphor P407 [0276] 52.8 mg of lyophilised FFS powder (FFSD017MC) was weighed into a tared 7 mL glass vial and 1.9 mL dH.sub.2O was pipetted into the vial. Vortex and sonication was used to dissolve/rehydrate the powder. 100 L of a 20% stock Kolliphor P407 solution was added by pipette to the vial and mixed by vortex, sonication was used to remove air bubbles. 400 L DMEM was added by pipette to the vial and mixed by vortex, sonication was used to remove air bubbles.

[0277] E 30 mM FFS in GrowDex (1/20 dilution) [0278] 52.8 mg of lyophilised FFS powder (FFSD017MC) was weighed into a tared 7 mL glass vial and 1.8 mL dH.sub.2O was pipetted into the vial. Vortex and sonication was used to dissolve/rehydrate the powder. 200 L of stock (as supplied 1.5% concentration) GrowDex solution was added by pipette to the vial and mixed by vortex, sonication was used to remove air bubbles. 400 L DMEM was added by pipette to the vial and mixed by vortex, sonication was used to remove air bubbles

[0279] All samples were stored in the fridge for 24 hours prior to viscosity measurement. Viscosity measurement was performed and the remainder of the samples were freeze dried. Following freeze drying all samples were used to prepare 1 mL of bio-ink in 1.5 mL Eppendorf tubes. These were left in the fridge overnight prior to repeating the viscosity measurements.

[0280] All bio-inks once prepared were observed to be pink in colour due to the phenol red in the DMEM media, indicating the pH of all materials to be similar and around neutral. On freeze drying all materials resulted in a fine powder that was observed to be white apart from the sample containing Kollifor P407 which had a pink tinge to it.

[0281] On rehydration of bio-inks with dH.sub.2O all bio-inks returned to pink and had their previous appearance. The bio-ink containing laminin sequence IKVAV (namely sample B) was initially able to be vortexed however quickly became viscous to the point that vortexing resulted in bubbles which were only partially able to be removed through sonication. Following immediate preparation of the samples they were visually observed as not being as viscous as they were following the initial 24 hour incubation in the fridge so they were returned to the fridge for overnight incubation. The flowing day it was noted that the bio-ink containing Kollifor P407 (namely sample D) remained as a flowing solution. Once this sample had been removed from the fridge and had reached room temperature it had returned to the set condition as prior to freeze drying.

[0282] As can be seen in the FIG. 14, the graph of the viscometry results, the viscometry of the bio-inks are comparable pre- and post-freeze drying. The only exception being the combination of Fmoc-FF/Fmoc-S and RGD which does shown a significant difference pre- and post-freeze drying, specifically that the gel is stiffer after freeze drying than prior to freeze drying. The increase in viscosity is also not a detrimental outcome as the viscometry value of 350 centipoise was still very much suitable for extrusion printing.

[0283] There was also a noticeable difference pre- and post-freeze drying with the combination of Fmoc-FF/Fmoc-S and IKVAV. In addition the bio-ink formed after freeze drying had a high variation in viscosity. It is likely that this is due to the difficulty when rehydrated the lyophilised powder and its high viscosity state.

[0284] The stability of the material over a longer time was then assessed. A 30 mM FFS bio-ink was prepared by the following method [0285] 52.8 mg of lyophilised FFS powder (FFS017MC) (i.e. a 1:1 mixture of Fmoc-FF and Fmoc-S) was weighed into a tared 7 mL glass vial and 2 mL dH.sub.2O was pipetted into the vial. Vortex and sonication was used to dissolve/rehydrate the powder. 200 L DMEM was added by pipette to the vial and mixed by vortex, sonication was used to remove air bubbles.

[0286] This material was stored at 4 C. for a three month period. The material was then removed from the fridge and allowed to come to room temperature before printing with a mechanical extrusion printer so as to form two rings one within the other. A small, 30 gauge, needle was used as the print head. Following printing the structure was immersed in DMEM to evaluate if it remained in the same construct or degraded.

[0287] As can be seen in FIG. 15 the material was printable and formed precise rings, one inside the other which required several layers to build up. Furthermore the construct remained stable following 24 hours immersion in DMEM.

[0288] Conclusion

[0289] A bio-ink has been developed that is printable from a single ink cartridge. Increasing the viscosity to a threshold of 250-300 Centipoise for extrusion printing was achievable through addition of DMEM as well as other components such as laminin sequences (IKVAV), fibronectin and Kolliphor P407. In addition it has been possible to prepare bio-inks and freeze dry them for reconstitution by an end user, providing a stable and convenient form for storage and transport. Such bio-inks are stable under refrigeration conditions for periods of several months and are stable once printed.

[0290] A Cellink Inkredible 3D printer was used to print three materials successfully to form structures which required additional layers of bio-ink to be added and for them to fuse together to form a single construct.

EXAMPLE 4

Drug Release from Hydrogels

[0291] We tested a hydrogel of the invention for its ability to provide slow or delayed release of drug.

[0292] Aim

[0293] To evaluate the ability of Fmoc-FF/S hydrogel to provide sustained release of a model drug compound. The drugs studied were Fluvastatin, Pravastatin, Propranolol and Sotalol.

[0294] Experimental

[0295] The 4 model compounds used in this study are shown below.

##STR00001##

[0296] Pre-Gel Preparation [0297] A 2 mM solution of each of the above compounds in distilled water was prepared, and then each solution was added to a vial containing 17.2 mg of Biogelx gel powder. [0298] The contents of each vial were alternately vortexed and sonicated for 30 secs-1 min to ensure the powder was completely dissolved and that a homogenous pre-gel solution was formed. [0299] The pre-gel solutions with drug compounds incorporated were stored at 4 C. until required. [0300] After overnight storage, the pre-gel solutions containing Fluvastatin, Pravastatin and Sotalol had become more viscous, but were still free flowing. The pre-gel solution containing Propranolol had become extremely viscous, and when the vial was inverted, the pre-gel was self-supporting (see results section for image). [0301] A 2 mM control solution of each drug in PBS solution (pH 7.4) was also prepared.

[0302] Release Assay [0303] The pre-gel solutions (containing drug compounds) were removed from cold storage, allowed to reach room temperature, and then vortexed and sonicated for 30 seconds to ensure a homogenous solution was achieved. [0304] Release buffer (PBS) and 24 well plates were pre-warmed in an incubator at 37 C. in a humidified atmosphere of 5% CO.sub.2. [0305] 500 L of release buffer added to 6 wells of the plate and 1.5 mL of distilled water was added to the surrounding wells. [0306] The plates were then equilibrated at 37 C. for 15 minutes. [0307] For each plate, 50 L of one of the pre-gel solutions containing a drug compound was added to a 24-well insert, and the insert placed in a well containing release buffer (performed in triplicate). [0308] 50 L of each of the 2 mM control solutions was added to a 24-well insert and added to a well containing release buffer in the appropriate plate (performed in triplicate). [0309] The plates were returned to the incubator for the required time. [0310] At each specified time point (see results section), the inserts containing the gel samples and the controls were moved to a fresh 24-well plate prepared in the manner described above. [0311] At each of the specified time points the release buffer remaining in the wells was transferred to HPLC vials and stored at 4 C. until HPLC analysis was performed.

[0312] HPLC Analysis

[0313] The samples of release buffer collected at each time point were analysed using

[0314] Drug Release Assay method at 220 nm.

[0315] An 1100 Series high-performance liquid chromatography pump (Agilent, USA) was used for the analysis which was performed using acetonitrile with 0.1% TFA and water with 0.1% TFA. The eluent solvent system had a linear gradient of 40% (v/v) acetonitrile in water for 3 mins, gradually rising to 80% (v/v) acetonitrile in water at 12 mins. This concentration was kept constant until 16 mins; after that the gradient was decreased, reaching 40% (v/v) acetonitrile in water at 19 mins. The data was then processed manually using the software Agilent Chemstation.

[0316] Results

[0317] Appearance of Pre-Gel Solutions

[0318] All solutions set to viscous gels. The propranolol gel seemed stiffest and retained its gel form even when inverted.

[0319] HPLC Analysis

[0320] The triplicate samples of release buffer were analysed by HPLC, and for the peaks corresponding to each model drug compound the peak area was measured. The average peak area was recorded, and using a standard curve prepared for each compound the concentration of each was determined.

[0321] Fluvastatin, Pravastatin and Sotalol

[0322] The data showed that for Fluvastatin, Pravastatin and Sotalol the release profile of the drugs from the hydrogel was slower than and the release time was extended compared to that from the control PBS solution. For propranolol both release time and profile altered significantly compared with the control and the propranolol-containing gel was identified for more detailed study.

[0323] Propranolol

[0324] The release profile for propranolol was examined in detail and found to be as follows in Table 5:

[0325] Retention time: 6.9-7.0 min

TABLE-US-00005 TABLE 5 Average Concentration peak area (ug/mL) Timepoint Control Gel Control Gel 30 min 5761.4 262.5 17.8 0.03 3 h 11536.5 2427.9 36.4 7.02 6 h 271.1 1920.2 0.06 5.38 9 h 11.9 1682.3 0 4.61 24 h 0 2661.3 0 7.78 48 h 0 2573.0 0 7.49 72 h 0 1498.7 0 4.02 5 d 0 1431.9 0 3.81 7 d 0 886.5 0 2.05

[0326] Graphs of the release of Propranolol and cumulative release of Propranolol are shown in FIGS. 16 and 17.

[0327] For Propranolol a sustained release from the gels over the whole 7-day study was hence observed and, lasting in excess of 7 days, was notably extended in time compared with the control.

[0328] Two further runs using propranolol were tested, differing only in that the concentrations of the peptides were 10 mM and 40 mM (rather than 20 mM in the example above). In both cases, significant drug concentration continued to be released after 72 hours and more, compared with the control in which drug release was below measurable limits by the 9 hour point.

[0329] Conclusion

[0330] The Fmoc-FF/S hydrogels were prepared containing a range of different drugs, giving clear, viscous and stable gels. Release of drug from these was extended compared with controls and significantly so in the case of propranolol.

[0331] Thus both examples demonstrated that extrusion of hydrogel precursors with cations can be used to produced 3D printed hydrogel structures and structures effective for slow/delayed release of drugs.

EXAMPLE 5

Further Drug Release from Hydrogels Study

[0332] Following the success of the first study set out above a further study was conducted using compounds.

[0333] Aim:

[0334] To evaluate the ability of Fmoc-FF/S hydrogels to provide sustained release of model compounds possessing specific structural features to gain a better understanding of the observed sustained release of previously screened drug Propranolol.

[0335] Experimental:

[0336] The 2 model compounds (Betaxolol and Quinidine) used in this study are shown below along with the previously studied Propranolol.

##STR00002##

[0337] Pre-Gel Preparation

[0338] Betaxolol Pre-Gel [0339] A 2 mM solution of each of the above compounds in distilled water was prepared, and then each solution was added to a vial containing 17.2 mg of Biogelx powder, namely lyophilized Fmoc-FF/Fmoc-S. [0340] The contents of each vial were alternately vortexed and sonicated for 30 secs-1 min to ensure the powder was completely dissolved and that a homogenous pre-gel solution was formed. [0341] The pre-gel solutions with drug compounds incorporated were stored at 4 C. until required. [0342] After overnight storage, the pre-gel solution containing Betaxolol had become significantly more viscous, and when the vial was inverted, the pre-gel was self-supporting (see results section for image). [0343] A 2 mM control solution of Betaxolol in PBS solution was also prepared.

[0344] Quinidine Pre-Gel [0345] A 2 mM solution of Quinidine in distilled water was prepared, and the solution was added to a vial containing 17.2 mg of Biogelx gel powder. A white suspension was formed. [0346] The contents of each vial were alternately vortexed and sonicated for several minutes, however the solid would not dissolve. [0347] The pH of the solution was adjusted using 0.5M NaOH. At approx. pH 10 a cloudy slightly viscous solution was formed. [0348] The pre-gel solution with drug compound incorporated was stored at 4 C. until required. [0349] After overnight storage, the pre-gel solution containing quinidine did not look significantly different, although the pH had fallen to approx. 9. [0350] A 2 mM control solution of Quinidine in PBS solution was also prepared.

[0351] Release Assay [0352] The pre-gel solutions (containing drug compounds) were removed from cold storage, allowed to reach room temperature, and the vortexed and sonicated for 30 seconds to ensure a homogenous solution was achieved. [0353] Release buffer (PBS) and 24 well plates were pre-warmed in an incubator at 37 C. in a humidified atmosphere of 5% CO.sub.2. [0354] 500 uL of release buffer added to 6 wells of the plate and 1.5 mL of distilled water was added to the surrounding wells. [0355] The plates were then equilibrated at 37 C. for 15 minutes. [0356] For each plate, 50 uL of one of the pre-gel solutions containing a drug compound was added to a 24-well insert, and the insert placed in a well containing release buffer (performed in triplicate). [0357] 50 uL of each of the 2 mM control solutions was added to a 24-well insert and added to a well containing release buffer in the appropriate plate (performed in triplicate). [0358] The plates were returned to the incubator for the required time. [0359] At each specified time point (see results section), the inserts containing the gel samples and the controls were moved to a fresh 24-well plate prepared in the manner described above.

[0360] At each of the specified time points the release buffer remaining in the wells was transferred to HPLC vials and stored at 4 C. until HPLC analysis was performed.

[0361] HPLC Analysis

[0362] The samples of release buffer collected at each time point were analysis using Drug Release Assay method at 220 nm.

[0363] An 1100 Series high-performance liquid chromatography pump (Agilent, USA) was used for the analysis which was performed using acetonitrile with 0.1% TFA and water with 0.1% TFA. The eluent solvent system had a linear gradient of 40% (v/v) acetonitrile in water for 3 mins, gradually rising to 80% (v/v) acetonitrile in water at 12 mins. This concentration was kept constant until 16 mins when the gradient was decreased to 40% (v/v) acetonitrile in water at 19 mins. The data was then processed manually using the software Agilent Chemstation.

[0364] Results:

[0365] Appearance of Pre-Gel Solutions

[0366] The Betaxolol containing solution set to a self-supporting gel, while Quinidine contain solution formed a viscous solution.

[0367] HPLC Analysis

[0368] The triplicate samples of release buffer were analysed by HPLC, and for the peaks corresponding to each model drug compound the peak area was measured. The average peak area was recorded, and using standard curve prepared for each compound the concentration of each was determined. Table and graphs of this data for each model compound can be seen below.

[0369] Betaxolol

[0370] The release time is set out in the Table 6 below, which as can be seen demonstrates an overall retention time of 7.4 min.

TABLE-US-00006 TABLE 6 Average Concentration peak area (ug/mL) Timepoint Control Gel Control Gel 30 min 1682.37 109.83 10.34 0.67 3 h 3685.33 1477.90 22.71 9.08 6 h 147.87 1037.33 0.90 6.37 9 h 12.10 486.83 0.07 2.98 24 h 0 839.17 0 5.15 48 h 0 244.40 0 1.49 72 h 0 44.37 0 0.27 5 d 0 17.43 0 0.11 7 d 0 0 0 0

[0371] Graphs of the release of Betaxolol and cumulative release of Betaxolol are shown in FIGS. 18 and 19.

[0372] Quinidine

[0373] The release time is set out in the Table 7 below, which as can be seen demonstrates an overall retention time of 8.5 min.

TABLE-US-00007 TABLE 7 Average Concentration peak area (ug/mL) Timepoint Control Gel Control Gel 30 min 3325.47 340.80 7.26 1.82 3 h 6122.67 581.77 32.25 3.10 6 h 86.37 622.87 0.47 3.32 9 h 7.77 421.20 0.04 2.25 24 h 0 960.23 0 5.11 48 h 0 1170.43 0 6.22 72 h 0 778.67 0 4.15 5 d 0 406.67 0 2.17 7 d 0 558.00 0 2.98

[0374] Graphs of the release of Quinidine and cumulative release of Quinidine are shown in FIGS. 20 and 21.

[0375] *It was noted that the results for the Quinidine Control corresponded to only 85% release.

[0376] Discussion

[0377] Comparing the data above, to the previously tested Propranolol (data shown below in FIGS. 16 and 17), it can be seen that both Betaxolol (FIGS. 18 and 19) and Quinidine (FIGS. 20 and 21) have a similar release profile to Propranolol.

[0378] Betaxolol was released significantly more slowly from the 10 mM Fmoc-FF/S gel than from the control insert (5 d vs. 9 h) however this release was over a shorter time than Propranolol, which showed a sustained release over the full 7-day study. Betaxolol, contains a similar amine chain to Propranolol, but only contains a single aromatic ring. The release results of Betaxolol indicate that although a conjugated aromatic system isn't necessary for sustained release of a molecule, the extent of aromaticity does influence the release, as Betaxolol with a single aromatic ring is release more quickly than Propranolol with a naphthalene system (2 rings).

[0379] Quinidine was also released significantly more slowly from the 10 mM Fmoc-FF/S gel than from the control, with sustained release being observed over the whole 7-day study, compared to 9 h for the control. HPLC analysis showed that 4.5% of the drug was still present in the remaining gel residue at the end of the experiment. Like Propranolol, Quinidine is a weakly basic compound with a conjugated aromatic system, but with a different chemotype. The release profile of this compound suggests that it is these general features which result in sustained release from the Fmoc-FF/S system, as opposed to specifically Propranolol-like structures.

[0380] Conclusion

[0381] It has been demonstrated that Fmoc-FF/S hydrogels provided steady, slow release rates of the additional model compounds, Betaxolol and Quinidine over simple diffusion. Both show a similar release profile to Propranolol, with sustained release of Quinidine occurring over the entire 7-day study and Betaxolol being released at a slightly faster rate (over 5 days). These results indicate that the extent of aromaticity within the encapsulated molecule may influence the release rate of the molecule, particularly when comparing the release of Propranolol and Betaxolol which possess similar chemotypes.

[0382] The invention hence provides 3D printing of peptide-containing hydrogels, and uses of those gels e.g. for cell growth and drug delivery.