WO2021038507A1

WO2021038507A1 - Enzymatically crosslinked silk fibroin hydrogel microfluidic platform, methods of production and uses thereof

Info

Publication number: WO2021038507A1
Application number: PCT/IB2020/058042
Authority: WO
Inventors: Mariana RODRIGUES DE CARVALHO; David CABALLERO VILA; Cristiana RODRIGUES DE CARVALHO; João BEBIANO COSTA; Viviana PINTO RIBEIRO; Joaquim Miguel Antunes De Oliveira; Subhas CHANDRA KUNDU; Rui Luís GONÇALVES DOS REIS
Original assignee: Association For The Advancement Of Tissue Engineering And Cell Based Technologies & Therapies (A4Tec) - Associação
Priority date: 2019-08-28
Filing date: 2020-08-28
Publication date: 2021-03-04

Abstract

The present disclosure relates to a transparent enzymatically crosslinked silk fibroin hydrogel-based microfluidic device that may be used ex vivo and in vivo, in tissue engineering applications, organ or tissue disease models, tissue-, organ- and body-on-a-chip, drug discovery, drug screening, tissue implant, tissue regeneration, and implantable microdevices. Namely, An enzymatically cross-linked silk fibroin hydrogel imprinted microfluidic device wherein the silk fibroin hydrogel retains its amorphous protein structure for at least 7 days; wherein said device is flexible and elastic; and wherein said microfluidic device comprises a microchannel configured to allow liquid media to flow, wherein the concentration of silk fibroin is from 3 % to 25 % (wt.%) of silk fibroin.

Description

D E S C R I P T I O N

ENZYMATICALLY CROSSLINKED SILK FIBROIN DEVICE, METHODS OF PRODUCTION AND USES THEREOF

TECHNICAL FIELD

[0001] The present disclosure relates to an implantable enzymatically crosslinked silk fibroin amorphous hydrogel-based microfluidic device and methods of producing said device.

[0002] The disclosed transparent enzymatically crosslinked silk fibroin hydrogel-based microfluidic device may be used, ex vivo and in vivo, in tissue engineering applications, organ or tissue disease models, tissue-, organ- and body-on-a-chip, drug discovery, drug screening, tissue implant, tissue regeneration, and implantable microdevices.

BACKGROUND

[0003] Biomedical research has increasingly moved towards the design, fabrication and implementation of microfluidic-based systems to efficiently improve point-of-care technologies such as drug discovery or drug screening, diagnostics, tissue engineering and regenerative medicine. The advent of microfluidic field has enabled precise fluidic manipulation and control of extremely small volumes. Due to its unique properties, poly(dimethylsiloxane) (PDMS) have typically been employed in the development of microfluidic systems. PDMS revolutionized traditional microfluidics which was previously based on silicon and glass. However, more biologically relevant, synthetic and natural hydrogel materials have recently been utilized in microfluidics fabrication to address the limitations of PDMS. PDMS has limited mechanical and biochemical properties. Typical hydrogels include collagen, gelatin, gelatin methacrylate (GelMA), agarose, alginate and other extracellular matrix proteins [1-4]. These hydrogel materials are cell-compatible, enabling better interfaces, namely with three-dimensional cell cultures.

[0004] Nevertheless, the mechanical properties of the existing hydrogel-based microfluidic materials such as their stiffness, only covers a limited range of native tissues, which limits their utility in mimicking the physiological conditions of the native tissue. Additionally, the degradability of the reported microfluidic hydrogel materials is far from desired. This is mainly because they are either not degradable or are only stable over a limited range of time frames thus challenging their use in tissue regeneration.

[0005] Microfluidic fabrication typically involves non-degradable materials including silicon and polydimethylsiloxane (PDMS) [5]. However, these devices are generally not implantable mainly because they are non-degradable, and can induce inflammation and trigger foreign body reaction. Strategies for developing more natural implantable systems comprise the use of agarose, gelatin, collagen and alginate [6-10] However, they do not present the best tuneable stiffness properties. They are not flexible enough to mimic the desired tissue. More recently, new biomaterials such as silk fibroin have been employed for tissue engineering applications. Recently, silk fibroin has been used for the development of primitive microfluidic systems. The combination of microfluidics and silk fibroin hydrogel offers a lot of advantages. Silk fibroin is FDA approved and has gained a lot of attention in the tissue engineering and regenerative medicine due to its excellent mechanical, biochemical, and cellular properties coupled with its biocompatibility, flexibility, degradation properties, water-based processing, and the presence of easily accessible chemical groups for functional modifications. This makes silk fibroin an ideal biomaterial for the development of implantable microfluidic systems [11, 12].

[0006] A novel class of enzymatically crosslinked silk fibroin hydrogels have recently been reported but their exploitation has mostly been limited to engineering in vitro 3D extracellular matrix (ECM) mimics for cancer research studies (e.g. cancer invasion, migration, proliferation, and others), bioinks, and as conduits for peripheral nerve regeneration applications [12, 13]. The horseradish peroxidase (HRP)/hydrogen peroxide (H2O2) cross-linking approach is used in polymers containing, or functionalized, with phenol group-containing molecules, including tyrosine, tyramine or aminophenol [14].

[0007] Very few works describing the use of silk fibroin as an implantable material have been reported in literature. Bettinger et al. [14] reported the fabrication of primitive microfluidic devices made by laminating water-stable micro-moulded silk fibroin membranes in b-sheet which were modified with macroscopic fluidic connections [15].

[0008] Document WO 2008/108838 A3 describes silk-based systems and devices such as microfluidic devices and methods for fabricating the same. However, in this document, silk solution is simply allowed to solidify on the moulds. No enzymatic crosslinking is employed to form a hydrogel. The resulting device is therefore in a film or layer of silk, resulting in b-Sheet conformation after initial processing.

[0009] Document US008975073B2 describes a microfluidic device comprising silk films coupled together to form a microchannel. However, it is described that the devices are fabricated by laminating micro-moulded and flat silk fibroin layers. Microfluidic layers are stacked, aligned, and bonded together at 70 °C, for 18 hours, under mechanical pressure. Additional 8% aqueous silk fibroin solution is used at the interface of the microfluidic layers. After the assembly was completed, the device was incubated in a 37 °C oven for 5 to 10 min to completely melt the gelatin, which was subsequently removed by flushing the channel with deionized water. The method described in this document is substantially different from the method disclosed in the present disclosure as the method disclosed in this document does not involve the use of silk fibroin solution at different concentrations and different ratios.

[0010] Document WO 2010/123945 A2 document describes silk fibroin hydrogels and uses thereof. This document discloses purified silk fibroin and method of purifying silk fibroins. This document also discloses hydrogels comprising silk fibroin with or without an amphiphilic peptide and methods for making hydrogels comprising silk fibroin.

[0011] Document WO18025186 describes enzymatic cross-linking of silk fibroin using horse radish and hydroxide peroxidase to obtain a hydrogel. However, the hydrogel obtained is only an intermediate step in the process of fabricating the final crystalline and tubular silk conduits disclosed. The silk fibroin hydrogel described in this document is therefore an intermediate product of nerve guidance conduits.

[0012] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure. GENERAL DESCRIPTION

[0013] The present disclosure relates to an implantable enzymatically crosslinked silk fibroin hydrogel-based microfluidic device and methods of producing said device.

[0014] The enzymatically crosslinked silk fibroin hydrogel-based microfluidic device of the present disclosure may be used in tissue engineering applications, organ or tissue disease models, drug discovery, drug screening, tissue implant, tissue regeneration or as implantable microdevices.

[0015] The present disclosure relates to a new methodology that comprises the use of an enzymatically cross-linked silk fibroin hydrogel for the microfabrication of a flexible, elastic and biodegradable 3D microfluidic chip with implantable characteristics. This methodology overcomes the problems of previously developed PDMS microfluidic platforms.

[0016] Specifically, this methodology overcomes the issues previously described by enzymatically cross-linking silkfibroin hydrogel, using horseradish peroxidase as enzyme and H2O2 as enzyme substrate to modify the silk fibroin solution into a hydrogel.

[0017] Silk is a naturally derived protein biomaterial with excellent biocompatibility and controllable degradation rates, thus suitable for tissue engineering and regenerative medicine applications. The new formulation is based on rapidly responsive silk fibroin hydrogels formed by a horseradish peroxidase (HRP) crosslinking reaction at physiological conditions, with potential use as an artificial biomimeticthree-dimensional (3D) matrix.

[0018] An advantage of using the method disclosed in the present disclosure is the ability to directly produce silk fibroin hydrogel microfluidic device in an amorphous state. This presents an opportunity to induce b-sheet conformation later, in many different ways, if necessary.

[0019] An aspect of the present disclosure comprises an enzymatically cross-linked silk fibroin hydrogel imprinted microfluidic device; wherein the silk fibroin hydrogel retains its amorphous protein structure for at least 7 days; wherein said device is flexible and elastic; and wherein said microfluidic device comprises a microchannel configured to allow liquid media to flow; wherein the concentration of silk fibroin is from 3 % to 25 % (wt.%) of silk fibroin.

[0020] In an embodiment, the concentration of silk fibroin is from 8 % to 20 % (wt.%) of silk fibroin, more preferably from 10 % to 12 % (wt.%) of silk fibroin.

[0021] In an embodiment, the enzymatic cross-linking of the silk fibroin hydrogel is by an enzyme horseradish peroxidase and an oxidizer hydrogen peroxide.

[0022] In a further embodiment, said peroxide amount ranges from 0.1 - 1 wt.%, preferably 0.2 - 0.4 wt.%. In another embodiment, the enzyme amount ranges from 0.5 - 1 mg/mL, preferably 0.7 - 0.84 mg/mL.

[0023] In an embodiment, the hydrogel is transparent for at least 7 days.

[0024] In an embodiment, the enzymatically cross-linked silk fibroin hydrogel imprinted microfluidic device is implantable.

[0025] In an embodiment, the device further comprises an extracellular matrix, a growth factor, a drug, a cell, and combinations thereof. In a further embodiment, the cell is selected from the list: cancer cell, stromal cell, immune system, endothelial cell, or combinations thereof.

[0026] In an embodiment, the enzymatically cross-linked silk fibroin hydrogel imprinted microfluidic device described in the present disclosure further comprises an inlet for liquid media to enter the microfluidic device; an intermediate meandering section for liquid media to flow; a first microchannel connecting the inlet and the intermediate meandering microchannel; an outlet for liquid media to exit the microfluidic device; a second microchannel connecting the outlet and the intermediate meandering part.

[0027] In an embodiment, the length of the device is from 5 mm to 150 mm, preferably 10 mm to 75 mm, more preferably 42 mm. In a further embodiment, the width of the inlet and the outlet are each from 200 pm - 10 mm, preferably 500 pm - 5 mm, more preferably 1 mm. In a yet further embodiment, the thickness of the intermediate meandering section is from 1 mm to 20 mm, preferably 2 mm to 10 mm, more preferably 6.5 mm. In another embodiment, the width of the microchannel is from 10 miti to 2 mm, preferably 100 pm to 1 mm, more preferably 200 pm.

[0028] In an aspect, the present disclosure relates to an enzymatically cross-linked silk fibroin hydrogel for use in medicine or veterinary, wherein the enzymatically cross- linked silk fibroin hydrogel is administrated in the form of an implantable imprinted microfluidic device. In an embodiment, said enzymatically cross-linked silk fibroin hydrogel administrated in the form of an implantable imprinted microfluidic device is for use in tissue engineering, organ disease models, tissue disease models, drug discovery, drug screening, tissue implant or tissue regeneration.

[0029] The present disclosure also relates to a kit comprising the implantable imprinted microfluidic device or the hydrogel administrated in the form of an implantable imprinted microfluidic device. In an embodiment, the kit further comprises at least one of the following components: Cells, growth factor, therapeutic agent, and mixtures thereof.

[0030] An aspect of the present disclosure comprises a method of producing the implantable imprinted microfluidic device, the method comprising the following steps: obtaining a polymethylsiloxane silanized master mould with predetermined microchannels; preparing an enzymatically cross-linked silk fibroin hydrogel with horseradish peroxidase and hydrogen peroxide; adding the silk fibroin hydrogel into the positive PDMS mould and incubate to form a hydrogel membrane.

[0031] In an embodiment, the method of producing the implantable imprinted microfluidic device further comprises the following steps: obtaining a master mould with predetermined microchannels, preferably a SU-8 master mould; adding a polymethylsiloxane (PDMS) solution, preferably at a ratio of 10:1 pre- polymercrosslinker, (Vpre-polymer/Vcrosslinker, and/or Wpre-polymer/Wcrosslinker), tO the master mold and allow the PDMS to cure in order to obtain a PDMS negative master mould which is a negative master mould replica; silanizing of the PDMS negative master mold to obtain a TCS-silanized negative PDMS master; pouring a second PDMS solution, preferably at a ratio of 10:1 pre-polymer:crosslinker (vpr_e-p_oiym_er/v_cr_ossiin_ker, and/or w_pre- p_oiym_er/w_cr_ossiin_ker) on top of the TCS-silanized negative PDMS master, degas, and cure; peeling off the negative PDMS mould to obtain a positive PDMS master mould; preparing an enzymatically cross-linked silk fibroin hydrogel with horseradish peroxidase and hydrogen peroxide; adding the silk fibroin hydrogel into the positive PDMS mould and incubate to form a hydrogel membrane.

[0032] In an embodiment, the aqueous silk fibroin solution has a silk fibroin concentration of at least 3 % (wt%), preferably from 3-25 % (wt%), more preferably from 8 to 20 % (wt%), more preferably from 10 to 12 % (wt%).

[0033] In another embodiment, the SU-8 master mould is produced using UV- photolithography.

[0034] In another embodiment, the PDMS is added to the SU-8 master mould and cured for at least 3 hours, preferably for a duration from 3 hours to 12 hours, at about 37 °C.

[0035] In another embodiment, the PDMS solution is allowed to cure on the TCS- silanized negative PDMS master for at least 3 hours, preferably for a duration from 3 hours to 12 hours, at about 37 °C, until the solution is completely cured.

[0036] In a yet another embodiment, the silk fibroin solution is added to the positive PDMS mould and incubated for at least 1 hour, preferably for a duration from 1 hour to 5 hours, at about 37 °C.

[0037] This horseradish peroxidase - H2O2 cross-linking approach allows the development of a substantially silk fibroin-based microfluidic device made of a flexible, implantable, biocompatible and biodegradable biomaterial. This hydrogel retains the amorphous protein structure for at least 7 days, allowing for cell encapsulation. Silk fibroin hydrogel resolution allows for the fabrication of micro-and nano-sized features, such as microchannels.

[0038] In an embodiment, the method of producing silk fibroin hydrogel-based microfluidic device comprises:

(i) the use of UV-photolithography for the fabrication of microfluidic channels;

(ii) replica moulding of the fabricated structures using a polymeric material (PDMS, and others such as polycarbonate (PC), polystyrene (PS), polyvinyl chloride (PVC), polyimide (PI);

(iii) TCS-silanization and second replica moulding of the microfluidic structures using a polymeric material (PDMS, and others); (iv) replication of the microfluidic structures using silk fibroin hydrogel;

(v) characterization of this process with respect to microstructural fidelity and cell viability.

[0039] In an embodiment, an initially produced aqueous silk solution is transformed into an amorphous and transparent hydrogel through a peroxidase-mediated cross-link reaction.

[0040] In an embodiment, silk fibroin was combined with horseradish peroxidase solution (HRP type VI, 0.84 mg/mL) and hydrogen peroxide solution (H2O2, 0.36 wt.%).

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0042] Figure 1 illustrates the process of producing the PDMS mould used to produce the enzymatically crosslinked silk fibroin hydrogel-based microfluidic device.

[0043] Figure 2 shows an embodiment of an ATR-FTIR spectra for the enzymatically crosslinked silk fibroin hydrogel (with 12 % concentration of silk fibroin) retaining the amorphous protein structure for at least 7 days. It is possible to visualize the structural change occurring from day 7: exhibition of a shift to a crystalline silk II structure as observed in peak shifts in amide I (1616.3 cm ¹) and amide II (1515.6 cm ¹) functionalities.

[0044] Figure 3 demonstrates the flexibility and elasticity of the transparent enzymatically crosslinked silk fibroin hydrogel at day 1 and at day 3.

[0045] Figure 4 schematically illustrates an exemplification of the structure of the enzymatically crosslinked silk fibroin microfluidic device. In this case, including a meandering serpentine channel, inlets and outlets. Figure 4A is without dimensions while Figure 4B is dimensions in pm.

[0046] Figure 5 shows SEM images of the 3D enzymatically crosslinked silk fibroin hydrogel structures after drying using critical-point drying. Scale bars: 500 pm (a and b), and 100 pm (c). [0047] Figure 6 schematically illustrates an example of what the enzymatically crosslinked silk fibroin hydrogel can be used for.

[0048] Figure 7 shows confocal microscopy images as described in the schematics of figure 6: endothelial cells seeded on the microchannel while colorectal cells are encapsulated throughout the enzymatically crosslinked silk fibroin microfluidic device.

[0049] Figure 8 shows the viability of cells encapsulated in the enzymatically crosslinked silk fibroin hydrogel-based microfluidic device. Live/dead assay was performed and observed under confocal microscope.

[0050] Figure 9 shows the perfusion of blue ink through the inlet (A); formation of soluble food colouring ink gradient (B); and diffusion of ink visible in the microchannels, allowing for the formation of diffusion gradients of drugs/nanoparticles (C).

[0051] Figure 10 shows liquid perfusion through the enzymatically crosslinked silk fibroin hydrogel-based microfluidic device's microchannel. (A) Shows perfusion of blue ink through the inlet. (Bl) and (B2) show magnified images at 0 and 5 seconds, respectively, showing liquid perfusion inside the serpentine microchannel (or meandering section). In an embodiment, a dynamic flow was achieved inside the microchip, as observed by the movement of impurity particles of the liquid along the microchannel (circles).

DETAILED DESCRIPTION

[0052] The present disclosure relates to an implantable enzymatically crosslinked silk fibroin microfluidic device and methods of producing said device.

[0053] The enzymatically crosslinked silk fibroin imprinted microfluidic device of the present disclosure may be used in tissue engineering applications, organ or tissue disease models, drug discovery, drug screening, tissue implant, tissue regeneration or as implantable microdevices.

[0054] An aspect of the present disclosure comprises an enzymatically cross-linked silk fibroin hydrogel imprinted microfluidic device; wherein the silk fibroin hydrogel retains its amorphous protein structure for at least 7 days; wherein said device is flexible and elastic; and wherein said microfluidic device comprises a microchannel configured to allow liquid media to flow; wherein the concentration of silk fibroin is from 3 % to 25 % (wt.%) of silk fibroin.

[0055] In an embodiment, the method for producing a flexible, elastic, biodegradable and implantable microfluidic device made substantially of enzymatically crosslinked silk fibroin hydrogel comprises the following:

Produce a SU-8 master mold with desired microchannels;

Add polymethy!siioxane (PDMS) solution at a ratio of 10:1 pre- polymercrosslinker (Vpre-polymer/Vcrosslinker, and/or Wpre-polymer/Wcrosslinker) tO the SU-

8 master mold and allow the PDMS to cure in order to obtain a PDMS mold which is a negative master replica of the SU-8 master mold;

Silanization of the PDMS negative master mold. This process is intended to passivate the PDMS surface to allow the fabrication of a second (positive) PDMS replica;

Pour PDMS solution at a ratio of 10:1 pre-polymer:crosslinker (v_pre- polymer/Vcrosslinker, and/or Wpre-polymer/Wcrosslinker) OP top Of the TCS-Silamzed negative PDMS master, degas, and cure until it is completely cured;

Peel off the negative PDMS mold to obtain a positive PDMS master mold; Prepare an aqueous silk fibroin solution with a concentration of at least 3 % (wt%), preferably from 5-25 % (wt%), more preferably 15-16 % (wt%);

Add horseradish peroxidase and hydrogen peroxide to the aqueous silk fibroin solution to form an enzymatically cross-linked silk fibroin hydrogel;

Add silk fibroin solution at the desired concentration into the positive PDMS mold and incubate to form a hydrogel membrane.

[0056] In an embodiment, the SU-8 master mould is produced using UV- photolithography.

[0057] In an embodiment, the SU-8 master mould is preferably produced on a silicon wafer.

[0058] In an embodiment, the PDMS is added to the SU-8 master mould and cured for at least 3 hours, preferably for a duration from 3 hours to 12 hours, at about 37 °C. [0059] In an embodiment, the salinization of the PDMS mould is by vapour phase method or aqueous method, preferably by trichloro(lH,lH,2H,2H-perfluorooctyl)silane (TCS) method. The purpose of the TCS layer is to aid the subsequent removal of the negative PDMS mould by preventing it from adhering to the first SU-8 master mould. In particular: place the PDMS mould in vacuum (e.g., desiccator) in the presence of a drop (drop volume from 5 pL to 100 pL) of pure TCS silanizing agent at room temperature. Afterwards, release from vacuum and store at about 70 °C for at least lh.

[0060] In an embodiment, the PDMS solution is allowed to cure on the TCS-silanized negative PDMS master for at least S hours, preferably for a duration from S hours to 12 hours, at about 37 °C, until the solution is completely cured.

[0061] In an embodiment, the silk fibroin solution is added to the positive PDMS mould and incubated for at least 1 hour, preferably for a duration from 1 hour to 5 hours, at about 37 °C.

[0062] In an embodiment, Figure 1 illustrates the process of producing the PDMS mould used to produce the silk fibroin hydrogel-based microfluidic device.

[0063] In an embodiment for better results, the thickness of the enzymatically crosslinked silk fibroin hydrogel layer can be controlled by controlling the volume of silk solution poured into the PDMS moulds.

[0064] In an embodiment, for better results, the mechanical properties of the silkfibroin hydrogel can be tuned by changing the concentration of silk fibroin proteins. In an embodiment, for better results, the silk fibroin hydrogel is enzymatically cross-linked with horseradish peroxidase and hydrogen peroxide, but other peroxidases and oxidizers may be used. In an embodiment, for better results, the silk hydrogel is functionalized with drugs or chemo-attractants.

[0065] In an embodiment, for better results, the hydrogel comprises from 3% to 25 % (wt %) of silk fibroin, preferably from 8% to 20 % (wt %) of silk fibroin, more preferably from 10% to 12 % (wt %) of silk fibroin. In a further embodiment, hydrogels comprising other silk fibroin concentrations compromises the microstructural fidelity of the microfluidic device. [0066] In an embodiment, an initially produced aqueous silk solution is transformed into an amorphous and transparent hydrogel, through a peroxidase-mediated cross-linking reaction.

[0067] In an embodiment, the enzymatically crosslinked silk fibroin hydrogel retains the amorphous protein structure for at least 7 days, allowing for cell encapsulation.

[0068] In an embodiment, Figure 2 shows an embodiment of an ATR-FTIR spectra for the 3D enzymatically crosslinked silk fibroin hydrogel (with 12 % concentration of silk fibroin) retaining the amorphous protein structure for at least 7 days. After this timepoint, a structural change occurs, with the exhibition of a shift to a crystalline silk II structure as observed in peak shifts in amide I (1616.3 cm ¹) and amide II (1515.6 cm ¹) functionalities.

[0069] In an embodiment, for better results, the enzymatically crosslinked silk fibroin microfluidic device further comprises a biological active agent, a therapeutic agent, an additive, a pharmaceutically acceptable excipient, a pharmaceutically acceptable carrier, and mixtures thereof.

[0070] In an embodiment, the enzymatically crosslinked silk fibroin hydrogel-based microfluidic device functions to mimic the extracellular matrices (ECM) of the body. For example, the silk fibroin hydrogel-based microfluidic device may serve as a physical support and/or an adhesive substrate for isolated cells during in vitro culture and subsequent implantation. As the transplanted cell populations grow and the cells function normally, the cells will begin to secrete their own ECM support and the silk- based microdevice may then biodegrade. The biodegradation of the silk fibroin hydrogel-based microfluidic device may be controlled by various manufacturing techniques known in the art.

[0071] In an embodiment, the enzymatically crosslinked silk fibroin hydrogel-based microfluidic devices are mechanically robust, transparent, flexible, elastic and possess microchannels with different geometries and sizes for seeding cells and flowing fluids (including body fluids) through the device.

[0072] In an embodiment, Figure 3 demonstrates the flexibility and elasticity of the transparent silk fibroin hydrogel at day 1 and at day 3. [007B] In an embodiment, Figure 4 schematically illustrates an exemplification of the structure of the enzymatically crosslinked silk fibroin microfluidic device. In this case, including a serpentine channel, inlets and outlets, without (A) and with (B) dimensions in pm.

[0074] In an embodiment, Figure 5 Scanning Electron Microscope (SEM) images of the 3D silk hydrogel structures after drying using critical point drying. Scale bars: 500 pm (a and b), and 100 pm (c).

[0075] In an embodiment, the enzymatically crosslinked silk fibroin hydrogel-based microfluidic devices are fabricated to support the growth of cells, including but not limited to eukaryotic cells (cancer cells, stromal cells, immune system, or endothelial cells). Other cell types could also be added as required.

[0076] In an embodiment, a portion of a surface of the microchannels in the silk fibroin hydrogel-based microfluidic device supports cell growth, in their native morphology.

[0077] In an embodiment, a portion of the enzymatically crosslinked silk fibroin hydrogel-based microfluidic device supports cell growth by encapsulating cells.

[0078] In an embodiment, Figure 6 schematically illustrates an example of what can be done using the hydrogel. For example, colorectal cancer microenvironment can be mimicked by seeding human colonic microvascular endothelial cells inside the microchannels, invading the matrix in response to VEGF gradients, while HCT-116 colorectal cells are encapsulated in the silk.

[0079] In an embodiment, Figure 7 shows the confocal microscopy images as described in the schematics of figure 6: endothelial cells seeded on the microchannel, while colorectal cells are encapsulated throughout the enzymatically crosslinked silk hydrogel microfluidic device.

[0080] In an embodiment, Figure 8 shows the viability of cells encapsulated in the enzymatically crosslinked silk fibroin hydrogel-based microfluidic device. Live/dead assay was performed and observed under confocal microscope. Live cells are stained in green, while dead cells are stained in red.

[0081] In an embodiment, Figure 9 shows the perfusion of blue inkthrough the inlet (A); formation of soluble ink gradient (B); and diffusion of food colouring ink visible in the microchannels, allowing for the formation of diffusion gradients of drugs/nanoparticles (C).

[0082] In an embodiment, oscillatory rheological measurements were acquired using a Rheometer (Kinexus Prot, Malvern) at 37°C with a plate-plate geometry. The rheological properties, namely the storage modulus and the loss modulus, were measured for the enzymatically cross-linked silk fibrin hydrogels at different concentration (6%, 12%, and 14 % of silk solution), as well as for PDMS gels to use as comparative data. The results were compared to PDMS, and are listed in Table 1. The rheological properties of the resulting hydrogels can be tuned by changing the concentration of silk, since hydrogels with higher silk concentration resulted in a greater storage modulus.

[0083] Table 1: Composition and rheological properties of hydrogels at a frequency of 0.1 Hz.

Incubation Storage modulus Loss modulus

Name Cells time (G', Pa) (G", Pa)

6% No 30min 19631151 30±16

12% No 30min 6585 ± 253 152 ± 48

14% No 30min 7173 ± 605 387 ± 64

PDMS No 8 hours 16794 ± 534 945 ± 51

[0084] In an embodiment, Figure 10 shows liquid perfusion through the enzymatically crosslinked silk fibroin hydrogel-based microfluidic device's microchannel. (A) Shows perfusion of blue ink through the inlet. (Bl) and (B2) show magnified images of liquid perfusion inside the serpentine microchannel.

[0085] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. [0086] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof.

[0087] The above described embodiments are combinable.

References

1. Buchanan, C.F., et al., Three-dimensional microfluidic collagen hydrogels for investigating flow-mediated tumor-endothelial signaling and vascular organization. Tissue engineering. Part C, Methods, 2014. 20(1): p. 64-75.

2. Liu, J., et al., Hydrogels for Engineering of Perfusable Vascular Networks. International journal of molecular sciences, 2015. 16(7): p. 15997-16016.

3. Johann, R. and P. Renaud, Microfluidic patterning of alginate hydrogels. Vol. 2. 2007. 73-9.

4. Lee, Y., et al., Photo-crosslinkable hydrogel-based 3D microfluidic culture device: Microfluidics and Miniaturization. Vol. 36. 2015.

5. Chen, C., et al., 3D-printed Microfluidic Devices: Fabrication, Advantages and Limitations-a Mini Review. Anal Methods, 2016. 8(31): p. 6005-6012.

6. Chung, B.G., et al., Microfluidic fabrication of microengineered hydrogels and their application in tissue engineering. Lab Chip, 2012. 12(1): p. 45-59.

7. Ling, Y., et al., A cell-laden microfluidic hydrogel. Lab on a Chip, 2007. 7(6): p. 756- 762.

8. Bertassoni, L.E., et al., Hydrogel bioprinted microchannel networks for vascularization of tissue engineering constructs. Lab on a Chip, 2014. 14(13): p. 2202- 2211.

9. Gillette, B.M., et al., In situ collagen assembly for integrating microfabricated three- dimensional cell-seeded matrices. Nat Mater, 2008. 7(8): p. 636-40.

10. Choi, N.W., et al., Microfluidic scaffolds for tissue engineering. Nat Mater, 2007. 6(11): p. 908-15.

11. Carvalho, M.R., et al., Microfluidics: Tuning Enzymatically Crosslinked Silk Fibroin Hydrogel Properties for the Development of a Colorectal Cancer Extravasation 3D Model on a Chip (Global Challenges 5-6/2018). Global Challenges, 2018. 2(5-6): p. 1870164.

12. Carvalho, C.R., et al., Tunable Enzymatically Cross-Linked Silk Fibroin Tubular Conduits for Guided Tissue Regeneration. 2018. 7(17): p. el800186.

13. Costa, J.B., et al., INKS FOR 3D PRINTING, METHODS OF PRODUCTION AND USES THEREOF. Patent WO/2018/225049.

14. Teixeira, L.S., et al., Enzyme-catalyzed crosslinkable hydrogels: emerging strategies for tissue engineering. Biomaterials, 2012. 33(5): p. 1281-90.

15. Bettinger, C.J., et al., Silk Fibroin Microfluidic Devices. Adv Mater, 2007. 19(5): p. 2847-2850.

Claims

C L A I M S

1. An enzymatically cross-linked silk fibroin hydrogel imprinted microfluidic device; wherein the silk fibroin hydrogel retains its amorphous protein structure for at least 7 days; wherein said device is flexible and elastic; and wherein said microfluidic device comprises a microchannel configured to allow liquid media to flow; wherein the concentration of silk fibroin is from 3 % to 25 % (wt.%) of silk fibroin.

2. The enzymatically cross-linked silk fibroin hydrogel imprinted microfluidic device according to the previous claim wherein the concentration of silk fibroin is from 8 % to 20 % (wt.%) of silk fibroin, more preferably from 10 % to 12 % (wt.%) of silk fibroin.

3. The enzymatically cross-linked silk fibroin hydrogel imprinted microfluidic device according to any of the previous claims wherein the enzymatic cross-linking of the silk fibroin hydrogel is by an enzyme horseradish peroxidase and an oxidizer hydrogen peroxide.

4. The enzymatically cross-linked silk fibroin hydrogel imprinted microfluidic device according to any of the previous claims wherein said peroxide amount ranges from 0.1 - 1 wt.%, preferably 0.2 - 0.4 wt.%.

5. The enzymatically cross-linked silk fibroin hydrogel imprinted microfluidic device according to any of the previous claims wherein the enzyme amount ranges from 0.5 - 1 mg/mL, preferably 0.7 - 0.84 mg/mL.

6. The enzymatically cross-linked silk fibroin hydrogel imprinted microfluidic device according to any of the previous claim wherein the hydrogel is transparent for at least 7 days.

7. The enzymatically cross-linked silk fibroin hydrogel imprinted microfluidic device according to any of the previous claims wherein the device is implantable.

8. The enzymatically cross-linked silk fibroin hydrogel imprinted microfluidic device according to any of the previous claims wherein the device further comprises an extracellular matrix, a growth factor, a drug, a cell, and combinations thereof.

9. The enzymatically cross-linked silk fibroin hydrogel imprinted microfluidic device according to any of the previous claim wherein the cell is selected from the list: cancer cell, stromal cell, immune system, endothelial cell, or combinations thereof.

10. The enzymatically cross-linked silk fibroin hydrogel imprinted microfluidic device according to any of the previous claims wherein the length of the device is from 5 mm to 150 mm, preferably 10 mm to 75 mm, more preferably 42 mm.

11. The enzymatically cross-linked silk fibroin hydrogel imprinted microfluidic device according to any of the previous claims further comprising: an inlet for liquid media to enter the microfluidic device; an intermediate meandering section for liquid media to flow; a first microchannel connecting the inlet and the intermediate meandering microchannel; an outlet for liquid media to exit the microfluidic device; a second microchannel connecting the outlet and the intermediate meandering part.

12. The enzymatically cross-linked silk fibroin hydrogel imprinted microfluidic device according to previous claims 11 wherein the width of the inlet and the outlet are each from 200 pm - 10 mm, preferably 500 pm - 5 mm, more preferably 1 mm.

13. The enzymatically cross-linked silk fibroin hydrogel imprinted microfluidic device according to the previous claims 11-12 wherein the thickness of the intermediate meandering section is from 1 mm to 20 mm, preferably 2 mm to 10 mm, more preferably 6.5 mm.

14. The enzymatically cross-linked silk fibroin hydrogel imprinted microfluidic device according to the previous claims 11-13 wherein the width of the microchannel is from 10 pm to 2 mm, preferably 100 pm to 1 mm, more preferably 200 pm.

15. An enzymatically cross-linked silk fibroin hydrogel for use in medicine or veterinary, wherein the enzymatically cross-linked silk fibroin hydrogel is administrated in the form of an implantable imprinted microfluidic device.

16. The enzymatically cross-linked silk fibroin hydrogel according to the previous claim for use in tissue engineering, organ disease models, tissue disease models, drug discovery, drug screening, tissue implant or tissue regeneration.

17. A kit comprising an implantable imprinted microfluidic device or a hydrogel according to any of the previous claims.

18. The kit or the hydrogel according to the previous claim wherein the kit further comprises at least one of the following components: Cells, growth factor, therapeutic agent, and mixtures thereof.

19. A method of producing the implantable imprinted microfluidic device according to any of the previous claims comprising the following steps: obtaining a polymethylsiloxane silanized master mould with predetermined microchannels; preparing an enzymatically cross-linked silk fibroin hydrogel with horseradish peroxidase and hydrogen peroxide; adding the silk fibroin hydrogel into the positive PDMS mould and incubate to form a hydrogel membrane.

20. The method of producing the implantable imprinted microfluidic device according to the previous claim further comprising the following steps: obtaining a master mould with predetermined microchannels, preferably a SU- 8 master mould; adding a polymethylsiloxane (PDMS) solution, preferably at a ratio of 10:1 pre- polymer CrOSSlinker, (Vpre-polymer/Vcrosslinker, and/or Wpre-polymer/Wcrosslinker), tO the master mold and allow the PDMS to cure in order to obtain a PDMS negative master mould which is a negative master mould replica; silanizing of the PDMS negative master mold to obtain a TCS-silanized negative PDMS master; pouring a second PDMS solution, preferably at a ratio of 10:1 pre- polymercrosslinker (Vpre-polymer/Vcrosslinker, and/or Wpre-polymer/Wcrosslinker) OP top Of the TCS-silanized negative PDMS master, degas, and cure ; peeling off the negative PDMS mould to obtain a positive PDMS master mould, preparing an enzymatically cross-linked silk fibroin hydrogel with horseradish peroxidase and hydrogen peroxide; adding the silk fibroin hydrogel into the positive PDMS mould and incubate to form a hydrogel membrane.

21. The method of producing the implantable imprinted microfluidic device according to any of the previous claims 19-20 wherein the aqueous silk fibroin solution has a silk fibroin concentration of at least S % (wt%), preferably from 3-25 % (wt%), more preferably from 8 to 20 % (wt%), more preferably from 10 to 12 % (wt%).

22. The method of producing the implantable enzymatically cross-linked silk fibroin imprinted microfluidic device according to any of the previous claims 19-21 wherein the SU-8 master mould is produced using UV-photolithography.

23. The method of producing the implantable enzymatically cross-linked silk fibroin imprinted microfluidic device according to any of the previous claims 19-22 wherein the PDMS is added to the SU-8 master mould and cured for at least 3 hours, preferably for a duration from 3 hours to 12 hours, at about 37 °C.

24. The method of producing the implantable enzymatically cross-linked silk fibroin imprinted microfluidic device according to any of the previous claims 19-23 wherein the PDMS solution is allowed to cure on the TCS-silanized negative PDMS master for at least 3 hours, preferably for a duration from 3 hours to 12 hours, at about 37 °C, until the solution is completely cured.

25. The method of producing the implantable enzymatically cross-linked silk fibroin imprinted microfluidic device according to any of the previous claims 19-24 wherein the silk fibroin solution is added to the positive PDMS mould and incubated for at least 1 hour, preferably for a duration from 1 hour to 5 hours, at about 37 °C.