CLEAVABLE COATING MATERIAL HAVING MICROBIAL FUNCTIONALITY

Abstract

Described is an object comprising a polymer coating, the coating comprising one or more polymers, wherein said polymers comprise a first cleavage site, and a first agent releasable from said coating upon cleavage of said first cleavage site. The first cleavage site is cleaved by a first compound specifically provided by a microbe belonging to a first group consisting of a limited number of microbial strains, species or genera, and not cleaved by any compound provided by any microbe not belonging to said first group, wherein cleavage of the said first cleavage site results in release of the said first agent from the coating, the release of said first agent being indicative for the presence of a microbe belonging to the said first group. Further, methods of detecting a microbial infection and of visualizing the presence of microbes are presented.

Claims

1. The article according to claim 8, wherein the coating further comprises: (c) one or more polymers that comprise a second cleavage site; and (d) a second agent releasable from the coating upon cleavage at the second cleavage site; wherein said cleavage at the second cleavage site is carried out by a second compound that is an enzyme specifically provided by a microbe that is a member of a second group consisting of a limited number of microbial strains, species or genera, but is not provided by any microbe not that is not a member of the second group, and wherein (i) the cleavage at the second cleavage site results in the release of the second agent from the coating, which release is indicative of the presence of a microbe belonging to the second group; (ii) the second cleavage site is different from the first cleavage site and is not cleavable by the first compound; and (iii) the limited number of microbial strains, species or genera of the second group are different from those of the first group; and (iv) the second agent is different from the first agent.

2. The article according to claim 1, wherein (a) the first agent is covalently bonded to the first polymer via a first linker, the first linker comprising the first cleavage site, and (b) the second agent is covalently bonded to a second polymer via a second linker, the second linker comprising the second cleavage site; and (c) optionally, the coating comprises (i) a first additional agent, which is released upon cleavage of the first cleavage site; and (ii) a second additional agent, which is released upon cleavage of the second cleavage site.

3. The article according to claim 2, wherein the first and/or second linker comprises one or more bonds selected from the group consisting of amide bonds, ester bonds, thioester bonds, and carbamate bonds.

4. The article according to claim 8, wherein the first agent is an antibiotic or a diagnostic agent.

5. The article according to claim 2, wherein the first and/or second additional agent is covalently bound to a first and/or second additional polymer via a first and/or second additional linker, the first and/or second additional linker comprising the first and/or second cleavage site respectively.

6. The article according to claim 2, wherein the first polymer, the first additional polymer, the second polymer and/or, the second additional polymer comprises a hydrogel.

7. The article according to claim 1, that is selected from the group consisting of a medical apparatus, a medical injection or infusion needle, and a medical implant.

8. An article coated with a polymer coating, comprising: (a) one or more polymers comprising a first cleavage site comprising an amino acid motif selected from the group consisting of PPTP (SEQ ID NO:2), PPSP (SEQ ID NO:3), LPATG (SEQ ID NO:4), LPETG (SEQ ID NO:5), LPDTG (SEQ ID NO:6), LPQTG (SEQ ID NO:7), NPQTN (SEQ ID NO:8), NPKTN (SEQ ID NO:9); and (b) a first agent releasable from the coating upon cleavage at the first cleavage site; wherein cleavage at the first cleavage site results in the release of the first agent from the coating.

9. The article according to claim 3, wherein the bonds are amide bonds.

10. The article according to claim 3, wherein the first and/or second linker comprises a peptide.

11. The article according to claim 1, wherein the first agent and/or the second agent is a therapeutic or a diagnostic agent.

12. The article according to claim 2, wherein the first agent, the second agent, the first additional agent or the second additional agent is a therapeutic or a diagnostic agent.

13. The article according to claim 4, wherein: (i) the antibiotic is selected from the group consisting of a -lactam antibiotic, a cephalosporin antibiotic, a macrolide, a cyclic depsipeptide and a tetracycline, and (ii) the diagnostic agent is selected from the group consisting of a fluorescent agent, a chemiluminescent agent, a bioluminescent agent and a radiation-emitting agent.

14. The article according to claim 17, wherein the antibiotic is vancomycin and the diagnostic agent is fluorescent agent IRDye800CW.

15. The article according to claim 5, wherein the first and/or second additional linker is identical to the first and/or second linker respectively, and the first and/or second additional polymer is identical to the first and/or second polymer, respectively.

16. The article of claim 13, further comprising a first additional agent releasable from the coating upon cleavage at a cleavage site having the same sequence as, but independent from, the first cleavage site from which the first agent is releasable.

17. The article of claim 16, wherein (i) the first agent is an antibiotic and the first additional agent is a diagnostic agent or (ii) the first agent is a diagnostic agent and the first additional agent is an antibiotic.

18. The article of claim 8, wherein the first cleavage site comprises LPETG (SEQ ID NO:5).

Description

[0155] The invention will be illustrated by the following non limiting examples and drawings, showing:

[0156] in FIG. 1, a schematic diagram of a coating according to the invention wherein an agent is covalently coupled to the polymeric matrix via linker molecules.

[0157] in FIG. 2 a schematic diagram of a coating according to the invention having a polymeric web with the agent embedded therein.

[0158] in FIG. 3, a schematic diagram of an object coated with a polymer coating according to the invention.

[0159] in FIG. 4, a schematic diagram of the object of FIG. 3 in the presence of a particular microbe that produces a compound specific for the cleavage site in the polymer coating.

[0160] in FIG. 5, a schematic diagram of the object of FIG. 4 upon irradiation.

[0161] in FIG. 6, a photograph of an orthopaedic plate coated with a polymer coating according to the invention, implanted in a human ankle, before closing of the wound.

[0162] in FIG. 7 a fluorescent image of the implant of FIG. 6 after closure upon irradiation by an external light source.

[0163] in FIG. 8 a photograph of an indwelling catheter coated with a polymer coating according to the invention, subcutaneously implanted in a human ankle.

[0164] in FIG. 9 a fluorescent image of the implant of FIG. 8 upon irradiation external light source.

[0165] in FIG. 10 a photograph of an orthopaedic plate and a catheter both coated and implanted in a human ankle (control implants).

[0166] in FIG. 11 a fluorescent image of the implants of FIG. 10 upon irradiation external light source.

[0167] In FIG. 1, a polymer, for example poly(ethylene glycol) is depicted by a solid line 11. Linker molecules, for example oligopeptides, depicted with dotted lines are covalently linked to the polymer. The linker molecules comprise a cleavage site, cleavable by one or more compounds, provided by one or more microbes. Said linkers 12 can e.g. be amino acid motifs comprising a specific portion of molecular structure, recognizable by e.g. a protease produced and excreted by a bacterium of group of bacteria. The linker is covalently bound to a first agent 13, e.g. a diagnostic agent such as a photon emitting agent, or a therapeutic agent, such as an antibiotic.

[0168] In FIG. 2, a polymer coating 21 is shown, which has a web structure. Within the web, an agent, such as a diagnostic or therapeutic agent 23 is entrapped. The agent is bound non-covalently in the polymer coating. The web structure comprises specific protease cleavage sites, cleavable by specific microbes. Cleavage will result in release of the agent 23 from the polymer coating 21.

[0169] In FIG. 3 a surface portion of an object 31, such as an implantable object or a working surface, is coated with a layer of polymer coating 32 of the invention comprising a first agent 33 (circles) and a second agent 34 (squares). Both first an second agents can be diagnostic molecules, preferably different from one another, i.e. giving a different signal upon release, or, accordingly, can be both a therapeutic agent. It is also possible for the first agent to be a diagnostic agent, and for the second agent to be a therapeutic agent, or vice versa. Both first an second agents can be embedded in the polymer as shown in FIG. 2, or be covalently linked to the polymer via linker molecules. In case the first agent and the second agent are both diagnostic agents differing in signalling upon release thereof from the coating, it is preferred to have these agents covalently linked to the polymer via different linker molecules. The first agent is in that case linked to the polymer via a first linker, and the second agent by a second linker. The first linker has a first cleavage site, different from the second cleavage site, present on the second linker. The first and second cleavage sites of the first and second linker, respectively, are preferably different from one another, so that different microbes, i.e. microbes that belong to two different groups as defined herein, each are capable of cleaving either the first or second cleavage site. Release of the first agent is indicative for the presence of a microbe belonging to the first group, and the release of the second agent is indicative for the presence of a microbe of the second group. By using different diagnostic agents, such as chemoilluminescent agents having different colour upon excitation with light of a defined wave length, for the first and second agents, a difference in colour is discriminative for the presence of a microbe belonging to the first or second group.

[0170] In FIG. 4, an object as in FIG. 3 is infected by microbes 41, such as pathogenic bacteria. In this case, the circles represent the first agent (circles) and can be a diagnostic, and the squares represent a first additional agent or a second agent , preferably being a therapeutic agent such a an antibiotic against the said microbe. Both first and second agent are coupled to the polymer of the coating via the same linker molecules, i.e. having the same cleavage site. In can be chosen to link both the first and the first additional agent to the very same first single linker molecule, so that both agents are released upon a single cleavage by a compound 42 (stars) of the microbe 41 (clouds). It can also be arranged such, that each linker molecule is only coupled to either the first agent or the first additional agent, and that a mixture of these differentially coupled linkers is coupled to the polymer of the coating. In both cases, both the first and first additional agents are released by cleavage by the same compound 42. Cleavage will result in release of the first (e.g. diagnostic) agent 33 and a first additional (e.g. therapeutic) agent 34. The infectious microbe can easily be detected upon infection by release of the diagnostic agent, whereas the infection is simultaneously be counteracted by the release of the therapeutic agent.

[0171] It can also be chosen to coat an object first with a first polymer coating comprising the first agent, followed by a second coating on the first coating, the second coating comprising the second agent or the first additional agent (or a combination thereof). In this arrangement, the infection by a microbe first arrives at the second coating, releasing the second agent, e.g. a diagnostic agent. In case the infection continues, it will arrive at the first coating, resulting in the first agent, such as a therapeutic like an antibiotic, to be released.

[0172] In FIG. 5, the infected object of FIG. 4 is irradiated with external light 51. Released first (diagnostic) agent (circles) is activated by the external light 51 and excites light of a different wavelength 52 as that of the external light 51. The said emitted light 52 can e.g. be near infrared light (NIRF). The irradiation makes the infection visible, while the released first additional agent (antibiotic) already acts to eliminate the pathogenic bacteria

[0173] In FIG. 6 a metallic orthopaedic device, coated with a coating of the invention is shown fixed onto bone in a human ankle, just before closing of the wound.

[0174] FIG. 9 shows an image taken on an IVIS (in vivo imaging station) camera at the site of the implant of FIG. 6 after infection with microbes and irradiation with an external light source. For further details, see Example 8. The emitted light indicates the presence of a microbial infection. FIG. 8 shows an indwelling catheter, subcutaneously arranged at a human ankle.

[0175] FIG. 9 shows an fluorescent image taken on an IVIS camera at the site of the implant of FIG. 8 after infection and irradiation with an external light source according to Example 8. The emitted light indicates the presence of a microbial infection.

[0176] FIG. 10 shows a photograph of an orthopaedic plate and a catheter implanted in a human ankle after closure of the wound.

[0177] FIG. 9 shows an fluorescent image taken on an IVIS camera at the site of the control implants of FIG. 8 after irradiation. The lack of an emitted signal is indicative for the lack of an infectious agent.

EXAMPLES

Example 1

Preparation of Polymeric Coatings

[0178] a) Synthesis of polymer.

[0179] The polymer PEGDA was synthesized using PEG(4000) or PEG(8000) and acryloyl chloride (Hem and Hubbell ,1998, J. Biomed. Mat. Res. 39(2):266-276).

b) Synthesis of linkers

[0180] Two peptide linkers were made using FMOC solid phase peptide chemistry (Fmoc Solid Phase Peptide Synthesis: A Practical Approach (Practical Approach Series), Chan and White, Oxford University Press, 1999)

[0181] i) CGGGLPETGGSGGK (SEQ NO:15) contains a first cleavage site, LPTEG, which is cleaved by Sortase A from S. aureus.

[0182] ii) CGGGAAFGGSGGK (SEQ NO:15) contains a second cleavage site, AAF, which is cleavable by serralysin from P. aeruginosa.

[0183] The peptides were cleaved from the resin using standard conditions (trifluoroacetic acid, 95%). Peptides were purified (>97% pure) by reverse phase high-performance liquid chromatography. Peptide structures can be confirmed by liquid chromatography-mass spectrometry.

c) Synthesis of a first agent and a first additional agent, IRDye800 covalently bound to vancomycin.

[0184] Vancomycin labeled with IRDye800CW was provided by LI-COR Biosciences (Lincoln Nebr., USA). Vancomycin-IRDye800CW (vanco-800CW) was synthesized by an adapted literature procedure. Vancomycin hydrochloride hydrate (3.0 mg, 2.0 mol) was added to a solution of IRDye800CW-NHS ester (1.0 mg, 0.86 mol) and N,Ndiisopropylethylamine (2.0 L, 11 mol) in dimethylsulfoxide (200 L). After overnight reaction at ambient temperature, the resulting bioconjugate was purified by reverse phase HPLC and lyophilized to afford a green flocculent solid (1.0 mg, 48%). Within the limits of HPLC detection, the final product did not contain unconjugated dye (detected at 780 nm) or unlabeled vancomycin (detected at 280 nm). UV/Vis (methanol) max=778 nm; Low Resolution Mass Spectrometry (ES/ammonium formate), m/z calculated for 2430.7 [M-H]-, found 810.6 [M-3H].sup.3.

d) Conjugate synthesis: first linker with a first cleavage sitefirst agent and first additional agent

[0185] i) A 1 g batch of the peptide linker as prepared above (step (b)(i)) was labeled on the the lysine (K) side chains with a first agent, IR dye 800CW NHS ester, (LI-COR Biosciences (Lincoln Nebr., USA)) using literature procedures (Tiyanont, K, et al. Proc. Natl. Acad. Sci. USA 2006, 103,11033-11038).

[0186] ii) A second 1 g batch of the peptide linker as prepared above (step (b)(i)) was labelled on the serine (S) side chains with a first additional agent, Vancomycin, (used as supplied by Sigma Aldrich) using standard coupling methods (Fmoc Solid Phase Peptide Synthesis: A Practical Approach (Practical Approach Series), Chan and White, Oxford University Press, 1999).

[0187] iii) A third 1 g batch of the peptide linker as prepared above (step (b)(i)) was labeled with both a first agent, IRDye800 and a first additional agent, vancomycin.

[0188] iv) A fourth 1 g batch of the peptide linker prepared above (step (b)(i)) was labeled with the product of step (c), the IRDye800-vancomycin covalent conjugate.

e) Synthesis of polymer-linker conjugates

[0189] The three peptide linkers as prepared above in steps (d)(i), (ii) and (iii) were each covalently bound to the PEGDA polymer synthesized in step (a) by adding the peptide linker (2 mM) to the PEGDA (8 mM-100 mM) in phosphate-buffered saline (pH 7.4) and stirring for 4 hours to overnight. Michael addition of the acrylate group to the sulfhydryl group on cysteine (Heggli, et al. (2003) Bioconjugate Chem. 14:967-973) was confirmed using Ellman's reagent (Pierce, Milwaukee, Wis.), thereby quantifying reduction in free sulfhydryl groups (Ellman (1959) Arch. Biochem. Biophys. 82:70-77). The solution was then polymerized using ammonium persulfate (APS, 20 mM) and N,N,N,N-tetramethylethylenediamine (TEMED, 51.6 mM) at 37 C. The products of the these coupling reactions are summarized in Table 1.

TABLE-US-00002 First addi- tional agent (cova- FirstLinker Firstagent lently Con- (firstcleavage (covalently bound jugate Polymer siteinbold) boundtoK) toS) 1e PEGDA.sub.4000 CGGGLPETGGSGGK IRDye800 (i) 1e PEGDA.sub.4000 CGGGLPETGGSGGK Vancomycin (ii) 1e PEGDA.sub.4000 CGGGLPETGGSGGK IRDye800 Vancomycin (iii) 1e PEGDA.sub.4000 CGGGLPETGGSGGK IRDye800- (iv) Vancomycin 1e PEGDA.sub.8000 CGGGLPETGGSGGK IRDye800 (vi) 1e PEGDA.sub.8000 CGGGLPETGGSGGK Vancomycin (vii) 1e PEGDA.sub.8000 CGGGLPETGGSGGK IRDye800 Vancomycin (viii) 1e PEGDA.sub.8000 CGGGLPETGGSGGK IRDye800- (ix) Vancomycin
f) Conjugate synthesis of a linker with a second cleavage sitesecond agent

[0190] A 1 g batch of the peptide linker as prepared above (step (b)(ii)) was labeled on the serine (S) side chains with a second agent, indocyanin green (LI-COR Biosciences (Lincoln Nebr., USA)) using literature procedures (Villaraza, A., et al. Bioconjugate Chem., 2010, 21 (12), pp 2305-2312) as shown in Table 2.

TABLE-US-00003 TABLE2 SecondLinker Second (secondcleavage Second Conjugate Polymer siteinbold) agent 1f(i) PEGDA.sub.4000 CGGGAAFGGSGGK indocyanin green 1f(ii) PEGDA.sub.8000 CGGGAAFGGSGGK indocyanin
g) Preparation of polymer coatings
Using the products from step (e) a number of different polymer coatings were prepared by simply mixing different proportions of conjugates 1 e (i)-(ix) and 1 f with each other.

[0191] A polymer coating (1 g (i)) was prepared that containing a polymer comprising a first cleavage site and a first agent (1 e (i)), and polymer comprising a first cleavage site and a first additional agent (1 e (ii)). In this way the first agent (IRDye800) and the first additional agent (vancomycin) can therefore be present in equal or different proportions, for example the said coating was prepared with 50% IRDye and 50% vancomycin.

[0192] Furthermore, any of conjugates 1 e (i)-(iv) can be mixed in with any of the conjugates 1 e (vi)-(ix). In this way a polymer coating (1 g (ii)) comprising 50% of a first polymer (PEGDA4000) comprising a first agent (IRDye800) (conjugate 1 e (i)) was mixed with 50% of a second polymer (PEGDA8000) comprising a first additional agent (vancomycin) (conjugate 1 e (vii)). The resultant polymer coating therefore had the advantage of containing a two types of PEGDA polymer so that the rheological properties of the coating that was made from the two polymers could be optimized.

[0193] By way of a further example a polymer coating (1 g (iii) was prepared that contained 50% of conjugate 1 e (i) and 50% of 1 f This polymer coating contained a first cleavage site, LPETG (SEQ NO:5), cleavable by sortase A from S. aureus so that IRDye800 can be released in the presence of S. aureus and a second cleavage site AAF, cleavable by serralysin from P. aeruginosa, so that indocyanin green can be released in the presence of P. aeruginosa. In this way the coating 1 f (v) can detect the presence of two specific microbes, namely S. aureus and P. aeruginosa.

[0194] Conjugates 1 e (iv) and 1 e (ix) enable the preparation of polymer coatings wherein the first agent and first additional agent are covalently bound to each other. In such a coating, the first and first additional agents are therefore released from the polymer coating at precisely the same location when a first cleavage site is cleaved by a compound specific for a microbe.

[0195] Different coatings can also be coated on an object in a subsequent manner, resulting in different coating layers as explained above.

Example 2

Coating Comprising an Agent Embedded in a Polymer Web

[0196] a) Synthesis of linker

[0197] A different linker to that used in Example 1 is necessary in order to synthesize a polymer coating where the linker is incorporated within the polymer matrix itself.

The linker (N.sub.3)GGGLPETGGSGGK(N.sub.3) was synthesized using Fmoc solid phase peptide synthesis as described above, using the modified amino acid building blocks (N.sub.3) and K(N.sub.3) as described in Van Dijk et al., Biomacromolecules, 2010, 11, 1608-1614.
b) Preparation of polymer-linker-polymer matrix

[0198] The polymer matrix wherein linkers comprising cleavage sites are incorporated within the hydrogel matrix itself was synthesized by the method of van Dijk, namely the Cu(I)-catalyzed 1,3-dipolar cycloaddition reaction between a cleavage site containing bis-azido peptide linker prepared in above in Example 2 step (a) and star-shaped alkyne-derivatized PEG moieties (Van Dijk et al, supra). The peptide linker contains the LPETG (SEQ NO:5) cleavage site that is cleaved by Sortase A from S. aureus.

c) Incorporation of first agent and first additional agent in a polymer matrix
i) The first agent (IRDye800), and the first additional agent (vancomycin) or the first agent-first additional agent conjugate as prepared in Example 1 step (c), were non-covalently incorporated into the hydrogel matrix by soaking in a solution of the polymer matrix. The products of these reactions are summarized in table 3. The products described in Table 3 can be used to prepare polymer coatings were the first and or first additional agents are therefore released from the polymer coating when sortase A, specific for the LPETG (SEQ NO:5) motif, cleaves the cleavage site present in the polymer matrix itself.

TABLE-US-00004 TABLE 3 Polymer matrix First agent First additional agent 2 c (i) IRDye800 2 c (ii) IRDye800 vancomycin 2 c (iii) IRDye800-vancomycin

Example 3

Preparation of a Coated Orthopedic Plate (FIG. 3)

[0199] Orthopaedic plates, screws and catheters were coated with the polymer coating with compositions according to step (g). For example a polymer coating 1 g (i) was coated onto orthopaedic plates, screws and catheters in a continuous process.

[0200] First a solution of the polymer prepared in Example 1 g (i) was made by dissolving the polymer coating in water and then the plate and catheter were drawn through the solution of the polymer coating at a rate of 1 to 2 meters/second into an infrared dying oven of approximately 1 meter in length at about 100 C. The objects were completely covered indicating the application of a substantially uniform coating. This process was done according to the standard procedures in U.S. Pat. No. 7,442,205, Stents and methods for preparing stents from wires having hydrogel coating layers thereon.

Example 4

Preparation of a Coated Working Surface

[0201] The coatings 1 f (i)-(iv) and 2 c (i)-(iii) were spray coated onto an operating table. By means The operating table was spray coated with coating 2 c (i). Briefly, the method comprised the following steps:

(a) grounding the surface of the medical device that is to be coated
(b) applying a coating 2 c (i), which further comprised a chloroform as a solvent, by (1) providing the nozzle apparatus comprising a chamber connected to at least one opening for dispensing the coating 2 c (i); (2) placing the coating into the chamber; (3) electrically charging the coating formulation; (4) creating droplets of the electrically charged coating formulation; and (5) depositing the droplets of coating formulation onto the grounded surface to form a coating on the surface.

Example 5

Culturing of S. aureus

[0202] A clinical isolate of Staphylococcus aureus was obtained with consent from a patient admitted to a general surgery ward. A culture of the clinical isolate was made. A 10 mL suspension of the isolate was prepared with a cell density of 510.sup.8 cell/ml culture.

Example 6

Preparation of Objects to be Implanted

[0203] a) Positive control
Orthopaedic plates, screws and a catheters as prepared in example 3 were incubated for 30 minutes in 10 mL of the isolate suspension as prepared in Example 5.
b) Negative control
i) Absence of S. aureus
An orthopaedic plate, two screws and a catheter were prepared according to Example 3. The coated objects were incubated in 10 mL of a solution of NaCl 0.9% (w/v) for 30 minutes. An overview of the prepared objects is shown in Table 4.

TABLE-US-00005 TABLE 4 Example S. aureus IRDye800 6 a Orthopaedic + + plate Screw + + Catheter + + 6 b Orthopaedic + plate Screw + Catheter +

Example 7

Implantation of Coated Implants

[0204] FIGS. 6 and 8 show, respectively, photographs of an orthopaedic plate with screws, and a catheter coated with a polymer coating according to the Example 6(a), that have been implanted in a human ankle. FIG. 10 shows a photograph of an orthopaedic plate, screws and a catheter coated with a polymer coating according to the Example 6 (b) that have been implanted in a human ankle, after closing of the implant wound.

[0205] The procedure for implantation of the objects coated in Example 6 (a) and (b), was carried out as followed:

[0206] The orthopaedic plates, screws and catheters were implanted in the ankle of a human cadaver. Implantation in a cadaver was used to simulate implantation of the said objects in a living patient. Each incubated plate was attached to the fibula with the respectively incubated screws, according to standard surgery procedures.

[0207] Each incubated catheter was inserted subcutaneously on the lateral side of the foot.

Example 8

Determining the Presence of Infection on an Orthopaedic Implant

[0208] Imaging of the objects implanted according to example 6 was performed 24 hours after implantation using an intra-operative clinical multispectral fluorescence camera (T3-imaging system, SurgOptix BV, Groningen, The Netherlands) and the IVIS Spectrum (excitation: 710 nm, emission: 800 nm, acquisition time 5 s, binning 4, F-stop 2, FOV 21.2).

[0209] The results obtained from the IVIS Spectrum and IVIS Lumina II were analyzed with Living Image 4.2. (Caliper LS, Hopkinton Mass., USA). Optimal detection limits were set to the lowest signal at which positive signal was effortlessly discriminated from negative controls. Signal intensity was determined by drawing regions of interests (ROI's) and measuring average counts in these regions. The signal was corrected for background by subtracting the background signal from the signal of interest, referred to in the text as net counts. A strong near infrared signal of 110.sup.54.210.sup.4 counts; p=0.007 was, attributed to IRDye 800CW.

[0210] As can be seen in FIG. 7, the coated orthopaedic plate and screws emitted a detectable signal, distinct from the background signal, in the presence of S. aureus. (white region in FIG. 7). FIG. 9 shows an image of a subcutaneously implanted catheter when infected with S aureus taken using a multi fluorescence camera.

[0211] The signals recorded in FIGS. 7 and 9 are indicative for the presence of S. aureus on or near the implant. S. aureus provides Sortase A, which is a compound specific for the LPETG (SEQ NO:5) motif contained in the peptide linker in the polymer coating. As described above, the linker was cleaved by the Sortase A compound and the IRDye800 was therefore released from polymer coating. Subsequently irradiation of the released IRDye800 by an infrared light induces photon emission from the dye which was recorded by the detector and can be seen in FIG. 9 as a white spot.

[0212] FIG. 11 shows an image of the control orthopaedic plate, screw and catheter prepared according to Example 6 (b), taken on a multispectral fluorescence camera. There is no intense white spot visible in the image, thus the coating does not emit a detectable signal indicating that the IRDye800 is still bound to the polymer coating and no S. aureus is therefore present.

Example 9

Detecting the Presence of Microbes on an Implanted Object

[0213] An implant comprising a polymer coating made from polymer conjugate 1 e (iv) prepared according to example 3, was implanted in a hind limb of immunocompetent mice (three in total). Three control mice received implants covered with the polymer conjugate 1 e (ii) that lacks the near infrared dye.

[0214] The six mice were subsequently injected with an inoculum of S. aureus strain Xen29, a standard, engineered strain of S. aureus that produces luciferase that facilitates localization of live bacteria by simultaneous imaging of luciferase bioluminescence and the fluorescence signal derived from IRDye800-vancomycin that is released from the polymer coating in the presence of S. aureus.

[0215] Three days after inoculation with S. aureus (Xen29), both bioluminescence and fluorescence imaging were performed with an IVIS Lumina II imaging system. A strong near-infrared fluorescence (NIRF) signal, attributed to IRDye800-vancomycin, appeared to co-localize with the bioluminescence signal (i.e. an intensity of 3.71059.8104 counts). No NIRF signal was observed in infected mice that received an implant coated with polymer lacking the IRDye800.

[0216] Removal of tissue surrounded the implant and visualising the tissue using a fluoresence detector yielded a target to background ratio of 4.2 for the implant coated with 1 e (iv) corrected for background signal (thus seen as a white spot as described above), over the control implant (coated with 1 e (ii)).

Example 10

Discrimination of the Presence of Microbes from a Sterile Foreign Body Reaction

[0217] To discriminate a microbial infection from a sterile foreign body reaction or aseptic inflammation, the fluorescence signal detected after intravenous injection with S. aureus (Xen29)-induced myositis (that is an immune response due to an infection by S. aureus, n=6) was compared to that in mice with sterile myositis induced by sterilely implanted Cytodex beads (n=6) (that is an immune response due to a foreign body and not an infection).

[0218] Three of the mice injected with S. aureus (Xen29) had myositis in the left hind limb, as well as contralateral sterile myositis in order to reliably compare signals within the same animal. Ex vivo, the muscle tissue with sterile inflammation showed fluorescent signals (1.81020.8102 counts) comparable to those of healthy tissue (1.2102 0.1102 counts, p=0.06, not significant). Again, muscle tissue infected with S. aureus (Xen29) emitted significantly higher fluorescence signals (4.11022.6102 counts) in comparison to non-infected contralateral healthy muscle tissue (p=0.008) and tissue with sterile inflammation (p=0.01). This example shows that the IRDye800 is only released in the presence of a compound provided by S. aureus and thus release of the IRDye from the polymer coating is indicative for the presence of S. aureus. The polymer coating prepared according to the invention can therefore be used to indicate the presence of microbes on or near the surface of the implanted object. The presence of sterile inflammation following bead implantation was confirmed by light microscopy using Giemsa staining, i.e., accumulation of leukocytes around the beads and absence of leukocytes in healthy muscle tissue. Leukocytes were also detected by anti-CD45 pan leukocyte fluorescence staining (data not shown). No bioluminescent bacteria were found in cultures from either the non-infected or sterilely inflamed muscle tissue.