FUNCTIONALIZED SET OF CARBON ATOMS IN THE SP2 HYBRIDIZATION STATE, A METHOD FOR PREPARING IT AND ITS USES, IN PARTICULAR FOR RENDERING A SURFACE ANTIBACTERIAL

Abstract

The present invention relates to a set of carbon atoms in the sp.sup.2 hybridization state functionalized with an antibacterial antibody or a lectin, and to an antibacterial peptide, a method for preparing it, and its uses, in particular for rendering a surface antibacterial. The present invention is also directed to a material covered with such a functionalized set, as well as to a method for preparing it.

Claims

1. A set of carbon atoms in the sp.sup.2 hybridization state, said set being in contact with: a plurality of compounds of the following formula (I):
(R-L.sub.1-W).sub.n—V-L.sub.2-X—Y  (I), in which: n is equal to 1, 2, or 3, in particular 3; V represents, for all n, —C(H).sub.3-n— or: when n is equal to 2, —C(H)═, C being in this case linked to L.sub.2 by a double bond; when n is equal to 1, —C≡, C being in this case linked to L.sub.2 by a triple bond; R is an aromatic hydrocarbon comprising from 2 to 6 condensed aromatic rings; L.sub.1 is a C.sub.1-C.sub.12 alkanediyl group; W represents a single bond, an arenediyl group, a heteroarenediyl group or a —O—Ar.sub.1— group wherein Ar.sub.1 is an arene or a heteroarene; L.sub.2 is a group of the following formula (A) -(L.sub.2a).sub.i-(L.sub.2b).sub.j- wherein: i and j are independently of each other selected from 0 and 1, with i+j=1 or 2; L.sub.2a is a C.sub.1-C.sub.12 alkanediyl, C.sub.2-C.sub.12 alkenediyl or C.sub.2-C.sub.12 alkynediyl group; L.sub.2b is a arenediyl group, a heteroarenediyl group or a —O—Ar.sub.2— group wherein Ar.sub.2 is an arene or a heteroarene; X is —C(═O)N—, —C(═O)O— or —C(═O)S— group; Y is an antibacterial antibody or a lectin; and a plurality of compounds of the following formula (II):
(R-L.sub.1-W).sub.n—V-L.sub.2-X—Z  (II), in which: R, L.sub.1, W, V, L.sub.2 and X are as defined above; Z is an antibacterial peptide.

2. The set according to claim 1, wherein said set of carbon atoms in the sp.sup.2 hybridization state is selected from graphene, graphene oxide, graphite, carbon nanotubes, fullerenes and fullerites.

3. The set according to claim 1, wherein: R is the 1-pyrenyl or the 2-pyrenyl; L.sub.1 is a C.sub.2-C.sub.8 alkanediyl group; and/or W represents a —O—Ar.sub.1— group wherein Ar.sub.1 is an arene or a heteroarene; and/or i and j are equal to 1; and/or L.sub.2a is a C.sub.2-C.sub.12 alkynediyl group; and/or L.sub.2b is an arenediyl group; and/or R, L.sub.1, W, V and G are as defined by the following formula: ##STR00003##

4. The set according to claim 1, wherein: Y is selected from the anti-Escherichia coli antibodies, the anti-gram positive bacteria antibodies, the anti-gram negative bacteria antibodies and the concanavalin A; and/or Z is selected from cecropins, defensins, magainins and dermaseptins.

5. The set according to claim 1, wherein the density of compounds of formulae (I) and (II) on the surface of the set of carbon atoms in the sp.sup.2 hybridization state is comprised from about 1 of these compounds per 300 nm.sup.2 area to about 1 of these compounds per 2 nm.sup.2 area.

6. The set according to claim 1, wherein: the molar ratio of the amount of compounds of formula (I) to that of compounds of formula (II) is comprised from 0.01 to 100; the set of carbon atoms in the sp.sup.2 hybridization state is graphene in the form of sheet, the molar ratio of the amount of compounds of formula (I) to that of compounds of formula (II) being comprised from 0.66 to 1.5, for example about 1.0; and/or the set of carbon atoms in the sp.sup.2 hybridization state is graphene in the form of flakes, the molar ratio of the amount of compounds of formula (I) to that of compounds of formula (II) being comprised from 0.33 to 0.66, for example about 0.5.

7. A method of preparing a set of carbon atoms in the sp.sup.2 hybridization state according to claim 1, comprising the following steps: (i) contacting a set of carbon atoms in the sp.sup.2 hybridization state with a compound of the following formula (III):
(R-L.sub.1-W).sub.n—V-L.sub.2-X-Q  (III), in which: R, L.sub.1, W, V and L.sub.2 are as defined in claim 1; X-Q is a group selected from —C(═O)O—N-succinimidyl, —C(═O)-halide —C(═O)—N.sub.3, —C(═O)—O—N-imidazolyl, —C(═O)—O—C(═O)—R′, with R′ being a linear, branched or cyclic C.sub.1-C.sub.12 alkyl, —C(═O)—O-catecholborane, —C(═O)—O-benzotriazole, to obtain a set of carbon atoms in the activated sp.sup.2 hybridization state; (ii) contacting the set of carbon atoms in the activated sp.sup.2 hybridization state as obtained in the previous step with a composition comprising an antibacterial antibody or a lectin and an antibacterial peptide, to obtain a functionalized set of carbon atoms in the sp.sup.2 hybridization state.

8. A surface antibacterial composition comprising the set of carbon atoms in the sp.sup.2 hybridization state according to claim 1.

9. A material comprising a substrate all or part of the surface of which is covered with a set of carbon atoms in the sp.sup.2 hybridization state in contact with compounds of formulae (I) and (II) according to claim 1.

10. The material according to claim 9, wherein said set of carbon atoms in the sp.sup.2 hybridization state is supported by a substrate.

11. A method for preparing a material according to claim 9, comprising the following steps: (i) contacting a set of carbon atoms in the sp.sup.2 hybridization state with a compound of the following formula (III):
(R-L.sub.1-W).sub.n—V-L.sub.2-X-Q  (III), in which: X-Q is a group selected from —C(═O)O—N-succinimidyl, —C(═O)-halide —C(═O)—N.sub.3, —C(═O)—O—N-imidazolyl, —C(═O)—O—C(═O)—R′, with R′ being a linear, branched or cyclic C.sub.1-C.sub.12 alkyl, —C(═O)—O-catecholborane, —C(═O)—O-benzotriazole, to obtain a set of carbon atoms in the activated sp.sup.2 hybridization state; (ii) contacting the set of carbon atoms in the activated sp.sup.2 hybridization state as obtained in the previous step with a composition comprising an antibacterial antibody or a lectin and an antibacterial peptide, to obtain a functionalized set of carbon atoms in the sp.sup.2 hybridization state; (iii) depositing the functionalized set of carbon atoms in the sp.sup.2 hybridization state as obtained in the previous step on the surface of a support to obtain said material.

12. A method for preparing a material according to claim 9, comprising the following steps: (i) contacting a set of carbon atoms in the sp.sup.2 hybridization state carried on the surface of a material with a compound of the following formula (III):
(R-L.sub.1-W).sub.n—V-L.sub.2-X-Q  (III), in which: X-Q is a group selected from —O—N-succinimidyl, -halide, —N.sub.3, —O—N-imidazolyl, —O—C(═O)—R′, with R′ being a linear, branched or cyclic C.sub.1-C.sub.12 alkyl, —O-catecholborane, —O-benzotriazole, to obtain an activated material; (ii) contacting the activated material as obtained in the previous step with a composition comprising an antibacterial antibody or a lectin and an antibacterial peptide, to obtain said material, functionalized.

Description

FIGURES

[0122] FIG. 1 shows optical images related to the growth of E. coli versus time, according to Example 2, with a surface such that the ratio of A:B is 0.35:0.65 (7:13).

[0123] FIG. 2 shows optical images related to the growth of E. coli versus time, according to Example 2, with a surface such that the ratio of A:B is 0.5:0.5 (1:1).

EXAMPLES

Example 1: Preparation of a Set of Carbon Atoms in the Sp.SUP.2 .Hybridization State Functionalized with an Antibacterial Antibody or a Lectin and an Antibacterial Peptide

[0124] A set of carbon atoms in the sp.sup.2 hybridization state, for example a graphene layer, is synthesized according to any technique well known to the person skilled in the art, in particular by CVD, then optionally deposited on a surface to be treated by any technique well known to the person skilled in the art, in particular by a liquid transfer method.

[0125] The graphene layer is soaked in a 1 μM solution in the tetrahydrofuran (THF) of a tripod of the following formula (as described for example by Mann et al., Angewandte Chemie International Edition 2013, 52, 3177-3180):

##STR00002##

[0126] and this, for one minute. The sample is then soaked in water in order to wash off the excess of THF. This results in a monolayer of tripods on the surface of the graphene with a density of about 1 tripod molecule per 2.7 nm.sup.2.

[0127] The resulting sample is incubated in a solution of PBS 1X. Anti-Escherichia coli antibodies (which have the ability to specifically bind Escherichia coli type bacteria) such as the LifeSpan BioSciences LS-058854, Abcam ab137967 or MyBioSource MBS568193 are then added to this solution, as well as molecules of cecropin (which have the particularity of creating holes in the cell membrane of bacteria and causing their cell death), such as cecropin A, cecropin B or cecropin P1 marketed by Sigma Aldrich (under the reference C6830 for cecropin A), in precise molar proportions, which are in particular discussed in the following example.

[0128] The tripod is designed in such a way that the N-Hydroxysuccinimide ester present on the top of the molecule will engage covalently bonds to any type of peptide or protein.

[0129] Thus, by injecting the antibody solution: cecropin into a stoichiometric A:B mixture, these molecules will covalently attach themselves one by one to the top of a tripod. After 30 minutes of incubation, there is an A:B ratio of antibodies and cecropin on the surface of the graphene.

[0130] Following the deposition of these molecules, the molecules ungrafted to a tripod are rinsed with a fresh solution of PBS1X (buffer solution) for 5 min.

Example 2: Evaluation of the Antibacterial Character of a Functionalized Set of Carbon Atoms in the Sp.SUP.2 .Hybridization State According to the Invention

[0131] Protocol

[0132] A graphene layer e.g. produced by CVD (Li et al., Journal of the American Chemical Society 2011, 133, 2816-2819) is deposited (e.g. according to Reina et al. Journal of the American Chemical Society 2011, 133, 17614-17617) on the surface of a microscope glass slide.

[0133] This graphene layer is functionalized according to the invention, in particular according to Example 1. Then, bacteria of Escherichia coli K12 type, which have been modified with a plasmid that allows them to synthesize the fluorescent protein Green Fluorescent Protein in addition to their metabolism (which allows the bacteria to be observed by fluorescence microscopy), are incubated on the surface for 30 min.

[0134] The bacteria that are not attached to the surface are then rinsed with PBS1X solution for 5 min. Then a nutrient solution for bacteria (Luria Bertani solution, also known as LB culture medium) is poured in. The temperature is maintained at 37° C. The bacteria attached to the surface of the graphene are thus, apart from the antibacterial surface, in conditions of exponential growth and proliferation.

[0135] Results

[0136] A control not part of the invention (surface with an A:B (antibody:antimicrobial peptide) ratio of 1:0) shows an exponential growth of the bacteria on the surface which starts to form a biofilm.

[0137] A:B Ratio from 0.25:0.75 to 0.40:0.60, in Particular 0.35:0.65 (7:13)

[0138] With such a ratio, it is observed that the functionalized layer of the invention causes not only the arrest of the growth of the bacteria on the surface but also their cell death. This is confirmed by the analysis of the fluorescence signal over time (FIG. 1). This signal decreases, which means that the cell membrane is damaged to such an extent that the GFP proteins leave the bacterium, which is therefore considered dead (or lysed).

[0139] Thus, such a ratio allows to obtain a bactericidal surface.

[0140] A:B Ratio Comprised from 0.40:0.60 to 0.60:0.40, in Particular 0.5:0.5 (1:1) With such a ratio, it is observed that the functionalized layer of the invention causes the bacteria to stop growing on the surface but the bacteria are not killed. Indeed, the signal related to the total GFP fluorescence intensity is constant over time (FIG. 2).

[0141] Thus, with this ratio, the layer still has a property of limiting the biofilm growth but the bacteria are no longer killed. It is therefore a bacteriostatic effect.