Implantable biocompatible reactor

Abstract

The invention concerns a bioreactor obtained by compressing a mixture of an enzyme, a conductor and chitosan. The conductor can consist of carbon nanotubes. This bioreactor can be produced according to the following steps: preparing a mixture of powders in which the proportion of enzyme powder relative to a carbon nanotube powder is of the order of 50/50 by weight; preparing a viscous solution of chitosan in a ratio of 5 to 15 (in mg) of chitosan to 0.75 to 1.25 (in ml) of acetic acid diluted to 0.4 to 0.6% by volume in water; adding the viscous chitosan to the mixture of powders in a proportion be weight of 3 to 5 of powder to 5 to 10 of chitosan; carrying out a first compression followed by light grinding; carrying out a second compression to produce a pellet; and drying at ambient temperature.

Claims

1. A bioreactor obtained by a method comprising the steps of: preparing a mixture of an enzyme powder and a carbon nanotube powder where the proportion of enzyme powder relative to carbon nanotube powder is of the order of 50/50 by weight; preparing a viscous solution of chitosan in a ratio of 5 to 15 (in mg) of chitosan to 0.75 to 1.25 (in ml) of acetic acid diluted to 0.4 to 0.6% by volume in water; adding the viscous chitosan to the mixture of enzyme powder and carbon nanotube powder in a proportion by weight of 3 to 5 of this mixture to 5 to 10 of chitosan; carrying out a first compression followed by light grinding; carrying out a second compression to produce a pellet; and drying at ambient temperature.

2. The bioreactor of claim 1, wherein the pressure applied during the first and the second compression is within a range from 2,000 to 6,000 kPa.

3. The bioreactor of claim 1, wherein the solution comprises from 0.002 to 0.005% in mass per volume of genipin.

4. The bioreactor of claim 1, wherein the solution comprises from 0.001 to 0.005% in mass per volume of caffeic acid.

5. The bioreactor of claim 1, wherein a porous chitosan-based membrane is laid on an active surface of the bioreactor and bonded at the periphery thereof.

6. The bioreactor of claim 5, wherein the porous chitosan-based membrane is obtained by a method comprising the steps of: preparing a solution in a ratio of 5 to 15 (in mg) of chitosan to 0.75 to 1.25 (in ml) of acetic acid diluted to 0.4 to 0.6% in water; stirring; pouring on a smooth support; and drying for a period from 2 to 4 days at ambient temperature.

7. The bioreactor of claim 5, having a pellet-shaped bioelectrode, wherein a conductor is glued by means of conductive glue to the surface of the pellet-shaped bioelectrode opposite to the active surface.

8. The bioreactor of claim 5, wherein the membrane comprises a plurality of pores having an average diameter in the range from 1 to 10 nanometers.

9. The bioreactor of claim 5, wherein the membrane comprises a smooth surface facing the pellet-shaped electrode and a rough surface facing outwards.

10. The bioreactor of claim 7, wherein the membrane comprises a plurality of pores having an average diameter in the range from 1 to 10 nanometers.

11. The bioreactor of claim 7, wherein the membrane comprises a smooth surface facing the pellet-shaped electrode and a rough surface facing outwards.

12. The bioreactor of claim 2, wherein the solution comprises from 0.002 to 0.005% in mass per volume of genipin.

13. The bioreactor of claim 2, wherein the solution comprises from 0.001 to 0.005% in mass per volume of caffeic acid.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The foregoing and other features and advantages will be discussed in detail in the following non-limiting description of specific embodiments in connection with the accompanying drawings, among which:

(2) FIG. 1 corresponds to FIG. 2 of European patent EP 2375481;

(3) FIGS. 2A and 2B respectively are a cross-section view and a top view of an embodiment of an electrode; and

(4) FIG. 3 shows current-vs.-time characteristics of various biofuels cells.

DETAILED DESCRIPTION

(5) If is here first provided to manufacture a bioelectrode pellet from a compression of chitosan, of an enzyme, and of a conductor, and possibly of a redox mediator and other additives, rather than from the compression of only one conductor and one enzyme. The conductor may advantageously be formed of multiwalled carbon nanotubes (MWCNT).

(6) A mixture of powders of an enzyme and of carbon nano-tubes, the proportion of enzyme powder relative to the carbon nanotube powder being in the order of 50/50 by weight, this proportion being likely to vary by approximately 20%.

(7) A viscous solution of chitosan is also prepared by adding chitosan powder in acetic acid diluted to 0.5% by volume heated up to 50 C., and by stirring for 2 hours at ambient temperature.

(8) The viscous chitosan is added to the powder mixture in a proportion by weight of 2 for the powder to 3 for the chitosan. For a chitosan pellet, 0.04 gram of powder and 0.06 gram of chitosan will for example be used.

(9) A mixture of the powder and of the chitosan in the viscous state is formed and a first compression, followed by a light grinding, is performed. A second compression which, like the first compression, is carried out at a pressure selected within a range from 2,000 to 6,000 kPa is then performed to provide a pellet, after which a drying is performed for from two to four days at ambient temperature (from 20 to 30 C.) so that the assembly can polymerize.

(10) A crosslinking agent for example, genipin at 0.0045% in mass per volume (g/100 ml) in the viscous chitosan solution alter 2 hrs of stirring. The genipin is previously solubilized in a solution of 12% of dimethylsulfoxide (DMSO) and 88% of water (H2O). To improve the resistance of the membrane to acids, a product such as caffeic acid by a proportion of 0.0032% in mass per volume (g/100 ml) in the viscous chitosan solution may be added to the initial mixture. The caffeic acid is previously solubilized to 4% in ethanol. The solution is stirred for 30 min before sampling 3 g to be spread on the smooth support for the drying, as previously described.

(11) A characteristic of the method described herein is that, during the second compression and the diving, the chitosan takes the form of long interconnected fibers having an approximate 30-nm diameter. It should be noted that a nanofiber and nanopore three-dimensional array is obtained simply by compression of the polymer with the powder and evaporation of the solvent at ambient temperature. As a result, the enzyme and the carbon nanotubes are immobilized in the chitosan fiber matrix and do not migrate to the outside of the pellet. This provides a significant advantage since the enzyme, which is trapped by the fiber matrix, remains protected and active for a long time. Further, the carbon nanotubes should be trapped, questions actually arising as to the possible noxiousness of carbon nanotubes in vivo.

(12) A biocathode comprising only one conductor and one enzyme, such as described in prior above-mentioned patent, has a 1-month stability in intermittent operation. A biocathode based on chitosan-MWCNT-laccase, such as described herein, has a stability greater than 2 months in continuous operation. In vitro measurements show that the stability in intermittent operation of the biocathode described herein exceeds six months. Further, such a stability is also ensured in vivo for a period longer than 200 days. This shows that the bioelectrode described herein provides the enzyme with an environment protecting the activity thereof, but also retains the enzyme within the electrode pellet and close to the carbon nanotubes for electric conduction. The porosity of the three-dimensional chitosan matrix allows a good diffusion of the enzyme substrates.

(13) As a variation, graphene, gold powder, or a conductive polymer such as polyaniline may be used as a conductor instead of using carbon nanotubes.

(14) Specific embodiments have teen described. Various alterations and modifications will occur to those skilled in the art. In particular, the polymer may be chitosan or another biocompatible polymer, for example: polyvinyl alcohol, poly(methylmethacrylate), gelatin, dextran, or copolymers such as chitosan-polyethylene glycol, or a mixture of these polymers.

(15) The method of manufacturing the electrodes based on conductive polymer and enzyme may be applied to the cathode or to the anode. Different enzymes may be immobilized in this structure: laccase, bilirubin-oxidase, polyphenol-oxidase, glucose-oxidase, glucose-deshydrogenase, catalase, peroxidase.

(16) According to another aspect of the present invention, a specific filtering membrane is provided for an enzyme bioelectrode such as hereabove or any other enzyme bioelectrode obtained by compression of a conductor, of an enzyme, and possibly of a redox mediatorthe redox mediator being optional in the cathode.

(17) As illustrated in FIGS. 2A and 2B, the electrode appears in the form of a pellet 20 for example having a circular shape in top view, a diameter from 0.5 to 1 cm, and a thickness from 0.5 to 2 mm. The lower surface of pellet 20 has a conductive strip 22 bonded thereto, for example, by conductive glue, for example a carbon paste 24, itself coated with a protective silicone glue layer 25, selected from among biocompatible glues. On the upper surface of pellet 20 is laidnot depositeda membrane 26 glued at its periphery to the pellet by a silicone glue ring 28.

(18) It is here provided to use a chitosan-based membrane for membrane 26. This membrane is for example obtained by starting from a solution in a ratio from 5 to 15, for example, 10 (mg) of chitosan to 0.75 to 1.25, for example, 1 (in ml) of acetic acid diluted to 0.4 to 0.6% by volume in water and heated up to 50 C. In a test, a dissolution of 200 mg of chitosan in 20 ml of acetic acid diluted to 0.5% by volume in water has been carried out. The mixture is stirred for two hours. 3 g of this mixture have then been sampled and spread on a non-adhesive smooth support (28-cm diameter), for example, an antistatic polystyrene cup, and dried for from 2 to 4 days at ambient temperature, for example, at a temperature from 20 to 30 C. In a test, the drying has been carried out for three days at 25 C.

(19) A flexible nanoporous film is thus obtained. Experiments carried out by the applicant have shown that this flexibility is due to the fact that the drying is carried out for a long time at ambient temperature. This characteristic is not obtained, for example, if drying temperatures greater than 40 C. ate used. For a film thickness in the range from 7 to 15 m, for example, 10 m, a porous membrane having average porous diameters in the range from 1 to 10 nanometers has been obtained. Conditions where this average diameter is in the range from 5 to 8 nm will be preferred to give way to glucose and to filter the compounds having the largest dimensions.

(20) In the same way as in the context of the forming of a pellet, a crosslinking agent, for example, genipin, and an agent of resistance to acids, for example, caffeic acids, may be added to the initial mixture.

(21) The obtained film exhibits a surface roughness difference between the two surfaces, which is due to the fact that one of the surfaces (the roughest) has been in contact with air rather than with the support (smooth surface). During the assembly, the rough surface will preferably be placed with its rough surface facing outwards with respect to the surface of the bioelectrode pellet. Indeed, the roughness difference on a thin film influences the ion diffusion and accordingly the electric resistivity. The inventors have shown that the chitosan film has a good ion conductivity (10-4 S.cm-1). Such an ion conductivity is better than that obtained with commercial membranes such as Nafion or cellulose acetate.

(22) The chitosan film described herein enables, by its mechanical properties, and particularly its flexibility and its adequate swelling ratio, to provide mechanical stability to the electrode by taking the shape of the surface of the pellet after swelling thereof in the liquid. It provides a biocompatible interface in contact with the tissues after implantation of the biofuel cell. It forms an efficient barrier against a possible salting out of the electrode components, oh the one hand, and against biological molecules originating from the extracellular fluid.

(23) The pore diameter may be adjusted by modifying: the chitosan concentration in the solvent (acetic acid), the chitosan/crosslinking agent ratio, the molecular weight of the powder chitosan placed in the initial acetic acid solution.

(24) FIG. 3 shows a characteristic in mA/ml (milliliters corresponding to the pellet volume) of current versus time in days for various biofuels cells. Curve A corresponds to the case where a cellulose acetate membrane has been used, curve B corresponds to the case a Nafion membrane has been used, and curve C corresponds to the cases where a chitosan-based membrane such as previously described has been used. It can be observed that the (negative) current is much greater in the case of the chitosan-based membrane and that the characteristics of the biofuels cell are actually not altered along time. It should further be noted that for cellulose acetate, the characteristic starts from a value of 0.15 and falls to a value in the order of 0.05 after approximately 70 days. With a Nafion membrane, a relatively constant characteristic is obtained, with current densities from two to three times smaller than with a chitosan-based membrane.

(25) Although the invention and the state of the art are described herein mainly in the case of a bioelectrode, it should be understood that the invention generally applies to any bioreactor implantable in vivo, such as defined at the beginning of the present description.