Implantable Device with Selective Cell Adhesion and Method of Production

Abstract

According to the present invention, an implantable device is provided, comprising a substrate on which at least one surface portion is provided. The chemical composition of the surface portion selectively enhances the cell-adhesion to the substrate.

Claims

1. An implantable device comprising: a substrate having a surface; wherein a first portion of the surface of the substrate, which is used for directly contacting biological cells or tissue, comprises: In.sub.(x)Ga.sub.(1-x)N, where x is in the range from 0.001 to 0.999, wherein the first portion promotes cell adhesion to the substrate and directs cell growth thereon, and further defines a semiconducting component of the device.

2. The device according to claim 1, wherein x is in the range from 0.01 to 0.88.

3. The device according to claim 1, wherein the first surface portion integrally covers the surface of the substrate.

4. The device according to claim 1, wherein the surface further comprises a second portion which is distinct from said first portion, wherein said second portion inhibits cell-adhesion to the substrate, and wherein said second portion comprises Indium nitride, InN.

5. The device according to claim 4, wherein said first and second portions integrally cover the surface of said substrate.

6. The device according to claim 1, wherein the substrate comprises Indium nitride, InN.

7. The device according to claim 4, wherein the first and/or second portion is provided as a thin layer on said surface of the substrate.

8. The device according to claim 4, wherein the first and/or second portion is provided as a thick layer on said surface of the substrate.

9. The device according to claim 1, wherein the first portion defines a pattern on the surface of the substrate, along which cell adhesion is enhanced.

10. The device according to claim 9, wherein said pattern comprises at least one line.

11. The device according to claim 9, wherein said pattern is delimited by a series of pillars comprising InN, arranged along at least one line.

12. The device according to claim 1, further comprising: at least one electrode for stimulating and/or monitoring contacted cells/tissues.

13. A method for selectively promoting the adhesion and growth of cells on a portion of a surface of a substrate of an implantable device, wherein said portion is used for directly contacting biological cells or tissue, the method comprising: forming a layer of In.sub.(x)Ga.sub.(1-x)N on said portion, where x is in the range from 0.001 to 0.999.

14. The method according to claim 13, further comprising: forming a first layer on InN on a substrate and subsequently selectively forming a second layer in In.sub.(x)Ga.sub.(1-x)N on top of said first layer said, where x is in the range from 0.001 to 0.999.

15. The method according to claim 13, further comprising: forming a first layer of In.sub.(x)Ga.sub.(1-x)N, where x is in the range from 0.001 to 0.999, on a substrate, subsequently forming a second layer of InN on top of said first layer, and subsequently selectively removing portions of said second layer, to expose the first layer of In.sub.(x)Ga.sub.(1-x)N.

16. The method according to claim 15, wherein said second layer covers said first layer.

17. The method according to claim 15, wherein said second layer of InN is selectively removed by etching.

18. The method according to claim 15, wherein said second layer of InN is selectively removed by ion beam milling.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Several embodiments of the present invention are illustrated by way of figures, which do not limit the scope of the invention, wherein:

[0039] FIGS. 1a to 1d schematically illustrate the structure of samples having a GaN, InN or In.sub.(x)Ga.sub.(1-x)N top layer respectively;

[0040] FIGS. 2a and 2b illustrate cell-viability results obtained using the samples of FIG. 1 for the cell lines MCF10A and HS-5 respectively;

[0041] FIGS. 3a, 3b and 3c illustrate cell adhesion results obtained using the samples of FIG. 1, wherein adhesion of different cell lines on the GaN, InN and In.sub.(x)Ga.sub.(1-x)N samples are shown in FIGS. 3a, 3b and 3c respectively;

[0042] FIG. 4 illustrates the morphology of HEK cells grown on different substrates;

[0043] FIG. 5 schematically illustrates a substrate of an implantable device according to a preferred embodiment of the invention;

[0044] FIG. 6 schematically illustrates a substrate of an implantable device according to a preferred embodiment of the invention.

[0045] FIGS. 7a and 7b schematically illustrate methods of further In.sub.(x)Ga.sub.(1-x)N functionalization;

[0046] FIG. 8a schematically illustrates a top view of a substrate of an implantable device according to a preferred embodiment of the invention;

[0047] FIGS. 8b and 8c schematically illustrate sides views of a substrate of an implantable device according to a preferred embodiment of the invention;

[0048] FIG. 9 shows directed growth of a human neuron cell on an implantable device having a substrate according to a preferred embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0049] This section describes the invention in further detail based on preferred embodiments and on the figures. First, the materials and methods for obtaining the presented results are described. It should be understood that technical features presented for a specific embodiment may be combined with features of other embodiments, unless specifically noted otherwise.

[0050] Materials and Methods

[0051] To simulate cell behavior in the presence of a device comprising a top layer, i.e. a cell-contacting layer, comprising In.sub.(x)G.sub.(1-x)N materials, different structures were synthesized by using Chemical Vapor Deposition on sapphire or silicon substrate. The structures are shown in FIG. 1a to 1d, wherein FIG. 1a shows a cell-contacting layer made of GaN, FIG. 1b, shows a cell-contacting layer made of InN. FIG. 1c shows a cell-contacting layer made of In.sub.(x)Ga.sub.(1-x)N, wherein 12.5% of Ga are present. FIG. 1d finally shows a cell-contacting layer made of In.sub.(x)Ga.sub.(1-x)N, wherein 25% of Ga are present. The top layer has a thickness of about 200 nm. The illustrated intermediate layers were chosen in order to optimize the transport of electrical signals from and to respective top layers. Their presence does not affect the results as to bio-adhesion and toxicity of the top layers. Cell-adhesion and -growth on Gallium nitride is known from the prior art. In order to assess the toxicity and the bio-adhesion of cells in presence of the respective top layer, Sigma XTT™ tests and bio-adhesion studies have been performed.

[0052] The experimental setup used for obtaining the reported results is described in what follows.

[0053] In order to perform adhesion microscopy, elements were placed in 24-well plates with 25 mm.sup.2 element per well. 500 μl of GFP(green fluorescent protein)-PC12 suspension containing 5′000 and 10′000 cells respectively in Dulbecco's Modified Eagle's Medium, DMEM −1% horse serum with nerve growth factor 100 ng/ml was added and incubated for 72 hours.

[0054] Before imaging on a Westburg Evos™ microscope, supernatants were aspirated and replaced with fresh DMEM medium without phenol red. Imaging of the elements was performed either by confocal microscopy experiments using a Zeiss LSM 510™ in fresh DMEM medium without phenol red, or by optical microscopy in reflective mode using a Zeiss™ microscope in phosphate-buffered saline, PBS.

[0055] Pfizer Adriblastina™ was used as Doxorubicin, Dox, ready-to-use injectable solution. Invitrogen™ NGF2.5S nerve growth factor was diluted in PBS/0.1% bovine serumalbumin and stored at −20° C.

[0056] Three cell lines were used in tests. First, the human mammary non-tumorigenic epithelial cell line MCF10A was grown in DMEM/F12 medium supplemented with 5% horse serum, epidermal growth factor EGF (Sigma-Aldrich™ 20 ng/ml), hydrocortisone (Sigma-Aldrich™ 0.5 mg/ml), cholera toxin (Gentaur™ 100 ng/ml), insulin (Sigma-Aldrich™ 10 mg/ml), 100 U/ml of penicillin and streptomycin.

[0057] Second, the HS-5 stromal cell line was cultivated in DMEM, 10% fetal bovine serum, 100 U/ml of penicillin and streptomycin.

[0058] Third, the PC-12 cell line derived from a transplantable rat neuroendocrine tumor of the adrenal medulla was grown in 10% horse serum, 5% fetal bovine serum, 100 U/ml of penicillin and streptomycin. Neuronal phenotype on this model is induced using nerve growth factor 100 ng/ml in DMEM −1% horse serum for 72 hours. PC12 cells stop dividing and terminally differentiate when treated with nerve growth factor. This makes PC12 cells useful as a model system for neuronal differentiation.

[0059] All cell lines were maintained at a temperature of 37° C. in an ambient environment containing 5% CO.sub.2 and 95% humidity.

[0060] Cell viability was determined using a colorimetric Sigma-Aldrich XTT™ assay in accordance with the manufacturer's instructions. 3′000 cells per well were plated on a 48-well plate with or without the different elements for 46 to 96 hours. Four hours before the end of the exposure, XTT was added. At the end of the exposure, optical densities of supernatants were read at 490 nm. A positive control of toxicity was obtained with doxorubicin treatment.

[0061] FIGS. 2a and 2b illustrate the viability of MCF10 mammary epithelial cells (FIG. 2a) and HS5 stromal cells (FIG. 2b) on respective cell-contacting layer of Sapphire, GaN, In.sub.(x)Ga.sub.(1-x)N (with 12.5% Ga and 25% Ga respectively) and InN. Cells were incubated for 48, 72 or 96 hours in presence of the different samples, including a control and dox sample. The latter is a positive control which induces cell death. Results are expressed in percentage (Optic Density, OD, calculated for a given sample/OD calculated for the control sample). As can be observed, the HS5 of MCF10 cell viability is not affected in the presence of In.sub.(x)G.sub.(1-x)N. Interestingly, the same is true for InN and Sapphire. The positive Dox control proves that the tested cells are not over-resistant.

[0062] FIGS. 3a, 3b and 3c illustrate the cell bioadhesion results obtained as described on the different GaN, InN and In.sub.(x)Ga.sub.(1-x)N layers. Besides HS5 and MCF10 cells, PC12 cells were tested as a model for neuronal cells. In the case of InN, substantially no cells are able to adhere on the substrate. In the petri dish, cells have grown on the plastic part as in the control sample, with no sign of induced toxicity. This confirms the viability results shown in FIGS. 2a and 2b. It has been observed that on In.sub.(x)Ga.sub.(1-x)N, the cell growth is similar to GaN, which is surprising due to the weak proportion of Ga in the InN alloy. For HS5 cells, the cell-growth has been observed to be clearly dependent on the proportion of Ga present in the InN alloy. Therefore it is possible to control the cell growth by varying the Ga ratio in In.sub.(x)Ga.sub.(1-x)N, i.e. by varying the proportion x.

[0063] The cell shape was controlled by using Human Embryonic Kidney, HEK, cells in confocal microscopy. FIG. 4 shows the morphology of the HEK cells on different samples. The HEK cells were transfected by the Green Fluorescent Protein, GFP, gene, which produces a fluorescent protein. The cell shape does not differ from the glass for GaN and In.sub.(x)G.sub.(1-x)N material, indicating a normal cell development on both substrates.

[0064] Neuronal cells are not able to produce collagen and can generally not stick to a glass/Saphhire substrate without precoating. Our results demonstrate the outstanding properties of InGaN which allows to promote not only adhesion but also neuronal network without any precoating.

[0065] Methods of In.sub.(x)G.sub.(1-x)N Functionalization:

[0066] Several methods to functionalize GaN are described in the literature. Similar methods can be applied by the skilled person for In.sub.(x)Ga.sub.(1-x)N surfaces. This allow the In.sub.(x)Ga.sub.(1-x)N surface to be biofunctionalized/bioconjugated with biomolecules or chemical functions for specific applications such as enhancing cell selectivity or promoting cell differentiation. One of these netgids is based on photochemical “functionalized-alkene” grafting (254 nm). This reaction requires to treat GaN by H plasma in order to get Ga—H bonding. Alternatively, GaN can be oxydated by a piranha solution and then coated by “functionalized silanes” (such as APTES). However these two techniques can induce an important bandgap modification. A method has been proposed which allows to get free amino groups on the GaN surface as well as keeping electronic properties. The method uses a radio frequency glow discharge plasma with humidified air to generate amino residues. These different techniques can be used in order to generate free amino groups on In.sub.(x)Ga.sub.(1-x)N/GaN surfaces (FIG. 7a). Amino groups can react with hetero bifunctional linkers which contain thiol reactive moieties (such as maleimide). Biomolecules which contain thiol function or carboxylic acid derivated can be easily conjugated by this way respectively on the maleimido-GaN layer (FIG. 7b) or on amino layer.

[0067] Applications:

[0068] In a preferred embodiment of the invention as illustrated in FIG. 5, the substrate 100 of an implantable device comprises surface 102 having a first portion 110, which comprises In.sub.(x)Ga.sub.(1-x)N, x being in the range from 0.001 to 0.999. The presence of the In.sub.(x)Ga.sub.(1-x)N, and the proportion x selectively enhances or promotes cell adhesion to the substrate 100.

[0069] Although the substrate 100 is depicted as being planar, it may be provided in any geometrical shape. The application in which the substrate is used generally defines the constraints on its shape, which the person skilled in the art will be capable of adapting appropriately without undue burden.

[0070] The portion 110 may cover the entire surface 102 or all surfaces of the substrate. Alternatively, the substrate itself may be provided as bulk In.sub.(x)Gn.sub.(1-x)N material, and regions of different x proportions may be provided thereon to selectively tune the cell-adhesion and cell-growth rates thereon.

[0071] According to another embodiment of the invention illustrated in FIG. 6, the surface 202 of the substrate 200 further comprises a second portion 220 which is distinct from said first portion 210. The second portion 220 inhibits cell-adhesion to the substrate 200, and comprises Indium nitride, InN. As both materials are non-toxic, the substrate may be used for in-vitro as well as in-vivo applications. For example, the substrate is usable for producing an implantable device which locally enhances the growth of cells, including neuronal cells.

[0072] Methods for forming thin (ranging from a thickness of a fraction of a nanometer to several micrometers) or thick layers as well as patterns of InN and/or In.sub.(x)Ga.sub.(1-x)N on a supporting substrate are as such known in the art and will not be described to any detail in the context of this invention. Specifically, a pattern of selectively cell adhesion enhancing paths or surfaces may be formed on an otherwise cell adhesion inhibiting substrate. To this end, a substrate made of InN may be locally doped with Ga along the paths or surfaces that form the pattern.

[0073] An exemplary substrate 300 is shown in a top view in FIG. 8a. A first cell adhesion promoting surface portion 310 comprises In.sub.(x)G.sub.(1-x)N, whereas a second cell adhesion inhibiting surface portion 320 comprises InN. Such a surface pattern may be obtained using different techniques. FIG. 8b is lateral view of showing a first example according to which the structure shown in FIG. 8a may be obtained. A first layer of InN 320 may be provided or formed on a substrate 300. The substrate may for example be made of Saphhire. In a further step, an addition layer of In.sub.(x)Ga.sub.(1-x)N if then formed on top of the layer of InN, in location corresponding to the desired surface pattern 310. Deposition methods, including for example Metal Organic Chemical Vapor Deposition, MOCVD, and using hard-masks, to achieve the formation of such layers, are as such known in the art.

[0074] FIG. 8c is a lateral view showing an alternative example according to which the structure shown in FIG. 8a may be obtained. A first layer of In.sub.(x)Ga.sub.(1-x)N, is provided on the supporting substrate 300, which corresponds to the Sapphire of the previous example. The first layer may have a thickness of 4000 nm, for example. A second layer of InN is formed on top of the first layer, substantially covering the first layer integrally. The thickness of the second layer is preferably less than the thickness of the first layer, and may for example be equal to about 200 nm. The desired surface pattern 310, to which contacted cells should adhere, is then formed by locally selectively removing the the thin layer of InN 320, thereby exposing the adhesion promoting layer of In.sub.(x)Ga.sub.(1-x)N situated there below.

[0075] According to one embodiment of the invention, the removal of the top InN layer from the so-formed stack is achieved by selective etching, using lithography methods known in the art.

[0076] According to an alternative embodiment, the removal of the top InN layer is achieved by direct milling using an ion beam. This method presents the advantage that patterns presenting fine ridges and grooves of the order of 1 micrometer and having a depth/height of about 2 micrometers are achievable.

[0077] Using the above outlined techniques, several surface patterns may be formed on a substrate of an implantable device, as shown in FIG. 9. The surface pattern may for example comprise surfaces of three-dimensional structures provided in/on the substrate. The pattern provided by the first portion 410 of the substrate 400, which comprises the In.sub.(x)Ga.sub.(1-x)N material and which is used to directly contact biological tissue, may for example be used to direct cell growth in a predetermined direction on the substrate of the implantable device. For example, a surface portion 410 of In.sub.(x)Ga.sub.(1-x)N may be locally delimited by pillars 420′ or guiding wall structures 420″ of InN, which are bio-compatible, but which do not promote cell-adhesion thereto. Cell growth of cells adhering to the first portion 410 is thereby promoting along the path delimited by the second portion 420, 420′, 420″, which imposes a physical limit to the cell-adhesion. The preferred direction of cell growth induced by the structures 420, 420′, 420″ is indicated by arrows.

[0078] FIG. 10 shows a corresponding experimental result, wherein a human primary neuron cell (highlighted on the figure) is shown adhering to a first substrate portion comprising of In.sub.(x)Ga.sub.(1-x)N. An axon of the neuron cell grows along the direction which is determined by the location of pillars of InN.

[0079] The results of FIG. 10 have been obtained according to the following protocol. To assess the behavior of primary neurons on patterned InGaN, GaN, or InN substrat, human primary neurons (Innoprot™, Spain) were seeded as per the manufacturer protocol at 125 000 cells/well in 48-well culture plate including patterned elements. The wells were coated with 1 ml of sterile 0.2% gelatin solution for 1 hour and washed 3 times with sterile water solution. The different elements were washed in ethanol, rinsed with PBS and set in the wells. 1 ml of cell suspension in serum free neuronal medium supplemented by neuronal growth supplement (Innoprot™, spain) and Penicillin/Streptomycin solution was added. The medium was changed to fresh supplemented medium the next day after establishing the culture and every 3 days thereafter. After 8 days of culture, elements were washed 3 times with PBS and fixed by using 2.5% Glutaraldehyde in Sodium Cacodilate for 1 h at room temperature. After 3 washes in sodium cacodilate, elements were dehydrated in ethanol (50%, 70% and absolute ethanol) and stored in 100% ethanol. To exclude a potential gelatin precoating effect related to the gelatin release in culture medium, the experiment was done with subculture of neuronal cells in 96-well plate without pre-coating on unpatterned elements. 23500 cells were seeded in 200 microliters of supplemented neuronal medium for 8 days and fixed as previously described and air dried. Elements have been finally visualized by using Scanning electron microscopy.

[0080] According to a further embodiment of the invention, an implantable electronic device is provided, which comprises a substrate presenting least one surface having a portion that comprises In.sub.(x)Ga.sub.(1-x)N, which is used for directly contacting biological cells or tissue. The surface portion comprising In.sub.(x)Ga.sub.(1-x)N defines a semiconducting component of the device, the band-gap of which is a function of the proportion of Ga in the In.sub.(x)Ga.sub.(1-x)N material. The sensitivity of the semiconducting component may therefore be tuned by adjusting the proportion of Ga.

[0081] The implantable device comprises signal processing means which are as such known by the person skilled in the art and which will not be detailed in further detail in the context of the present description. The signal processing means may comprise means for amplifying or filtering a signal received from/transmitted by the semiconducting component. The signal processing means are operatively connected to the semiconducting component implemented by the In.sub.(x)Ga.sub.(1-x)N surface portion, which directly contacts the biological tissue.

[0082] The In.sub.(x)Ga.sub.(1-x)N portion of the substrate surface may therefore be used to transmit a signal emitted by the cell or tissue to the signal processing means or to transmit a processed signal to the cell or tissue. In the latter case, the implantable device may further comprise signal generating means such as a tunable voltage or current source and appropriate controlling/processing means as an input to the signal processing path. Such means are as such known to the skilled person.

[0083] In a preferred embodiment, the implantable device comprises at least one electrode, preferably arranged at an edge thereof, for contacting and selectively stimulating and/or monitoring contacted cells/tissues.

[0084] The implantable device may further comprise functionalized semiconductors such as a field effect transistors, FET, or single electrode transistors, SET, for processing or transmitting a received signal to or from the implantable device. The described components are preferably provided on a common substrate of the implantable device. In a preferred embodiment, the implantable device may be an in-vivo component of a Brain-Machine interface.

[0085] It should be understood that the detailed description of specific preferred embodiments is given by way of illustration only, since various changes and modifications within the scope of the invention will be apparent to the skilled person. Unless specified otherwise, features of a described embodiment of the invention may be combined with features described in the context of other embodiments. The scope of protection is defined by the following set of claims.

REFERENCES

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