Surface-modified polymeric substrates grafted with a properties-imparting compound using clip chemistry

Abstract

The present invention relates to an efficient method for grafting a properties-imparting compound onto a polymeric substrate containing carbon-hydrogen (C—H) bonds using clip chemistry. The method of the invention includes coating the substrate with the properties-imparting compound and irradiating it with a reactive light source, and repeating this sequence at least once. The present invention further relates to surface-modified polymeric substrates grafted with a properties-imparting compound, in particular obtained with the method of the invention, medical devices comprising same, and non-medical of said surface-modified polymeric substrates.

Claims

1. Method for grafting a properties-imparting compound onto a polymeric substrate containing carbon-hydrogen (C—H) bonds, said method comprising: a) providing a polymeric substrate; b) coating the polymeric substrate with a properties-imparting compound comprising a photoactive aryl-azide moiety of formula (I): ##STR00060## with X.sub.1, X.sub.2, X.sub.3 and X.sub.4 independently representing a hydrogen or a fluorine atom, a C.sub.1-C.sub.6 alkyl group, NO.sub.2 or OH, and L representing NH, —C(O)O—, —S—, —C(O)NH—, —NHC(O)—, —OC(O)—, —NHC(O)NH—, —NHC(S)NH—, —C(O)NRC(O)— with R representing a C.sub.1-C.sub.6alkyl or triazolyl, so as to obtain a homogeneous dry layer of said properties-imparting compound coated on at least part of the polymeric substrate, and to bring said aryl-azide moiety of formula (I) into covalent bonding proximity with the carbon-hydrogen bonds of the polymeric substrate, provided that when the properties-imparting compound is a polymer, it is a block- or gradient-copolymer with a block or a region rich in repeated units A, said repeated units A comprising the aryl-azide moiety of formula (I) as defined above; c) irradiating the coated polymeric substrate with a reactive light source, for a time t.sub.1 sufficient to form nitrenes that undergo insertion reactions into carbon-hydrogen bonds of the polymeric substrate, t.sub.1 being equal to or less than 30 minutes, thereby yielding a grafted polymeric substrate; d) optionally washing the obtained grafted polymeric substrate; e) repeating steps b), c) and optionally d) at least once; and f) optionally drying the grafted polymeric substrate obtained at the end of step e), said properties-imparting compound providing anti-fouling properties, antibacterial properties, or rendering the polymeric substrate radio-opaque or visible in medical imaging; and wherein the method is carried out directly on the polymeric substrate without any prior treatment step.

2. The method of claim 1, wherein the polymeric substrate is a polymeric implantable substrate.

3. The method of claim 1, wherein the polymeric substrate is selected from aliphatic polyesters and copolyesters, copolymers of aliphatic polyesters and polyethers, polycarbonate, polydioxanone, polypropylene, polyethylene, polyethylene terephthalate, polyethylene oxide, polyurea, poloxamer, poloxamine, silicone, polycarboxylate, polyether ether ketone, ABS, Polystyerene, Polyvinylchloride, and polyacrylates.

4. The method of claim 1, wherein the properties-imparting compound is a hydrophilic block-copolymer with a block comprising repeated units A or a gradient-copolymer with a block a region comprising repeated units A, said repeated units A comprising the photoactive aryl-azide moiety of formula (I) as defined in claim 1 with the nitrogen atom covalently bound to the surface of the surface-modified polymeric substrate, said hydrophilic block- or gradient-copolymer imparting anti-fouling properties.

5. The method of claim 4, wherein the hydrophilic block- or gradient-copolymer contains at least repeated units A and B, wherein Repeated units A comprise the photoactive aryl-azide moiety of formula (I) as defined in claim 1, and Repeated units B lack the photoactive aryl-azide moiety of formula (I) as defined in claim 1.

6. The method of claim 5, wherein the molar ratio of repeated units A over repeated units B (repeated units A/repeated units B) is of between 0.01% and 50.

7. The method of claim 4, wherein the hydrophilic block- or gradient-copolymer is a poly(ethylene glycol), a poly(ethylene oxide), a poly((meth)acrylatePEG), poly((C.sub.1-C.sub.6)alkylamino(meth)acrylate), a linear poly(quaternary ammonium) with a molecular weight of less than 20 000 g.Math.mol.sup.−1, a zwitterionic poly(betaine), a poly(vinylpyrrolidone), a polylysine, a polyoxazoline, a polyoxazine, a polysarcosine block- or gradient-copolymer or a polyoxazoline-polysarcosine block-copolymer or a polyoxazoline-polyoxazine copolymer.

8. The method of claim 1, wherein the properties-imparting compound is an antibacterial agent selected from: a polymer selected from: a quaternized poly(vinylpyridine), a quaternized poly(dimethylaminoethylacrylate), a linear quaternized poly(ethyleneimine), a polylysine, a quaternized polylysine, a copolyester of quaternized poly(5-Amino-δ-valerolactone), a quaternized polyoxazoline-polyethyleneimine copolymer, wherein the quaternized polymers are quaternized with a C.sub.3-C.sub.15 alkyl, a quaternary ammonium of formula (IIa): ##STR00061## with X.sub.1 to X.sub.4 and L as defined above, q an integer from between 0 and 10, r an integer from between 0 and 3000, and R.sup.1 and R.sup.2 each independently selected from a hydrogen atom or a C.sub.1-C.sub.6 alkyl group, R.sup.3 independently selected from a hydrogen atom or a C.sub.1-C.sub.9 alkyl group and B— representing a pharmaceutically acceptable anion; a quaternary phosphonium of formula (IIb): ##STR00062## with X.sub.1 to X.sub.4 and L as defined above, q an integer from between 0 and 10, r an integer from between 0 and 3000, R.sup.1, R.sup.2 each independently selected from a hydrogen atom or a C.sub.1-C.sub.6 alkyl group, R.sup.3 independently selected from a hydrogen atom or a C.sub.1-C.sub.9 alkyl group, and B— representing a pharmaceutically acceptable anion; a quaternary pyridinium of formula (IIc) ##STR00063## with X.sub.1 to X.sub.4 and L as defined above, q an integer from between 0 and 10, r an integer from between 0 and 3000, R selected from a hydrogen atom or a C.sub.1-C.sub.9 alkyl group, and B— representing a pharmaceutically acceptable anion; an antibacterial peptide of 25 amino-acids or less, comprising a pending group of formula (I) as defined in claim 1.

9. The method of claim 1, wherein the properties-imparting compound is a radio-opaque iodinated contrast agent, a gadolinium complex, a fluorescent compound, or a near-infrared fluorescent compound.

10. The method of claim 9, wherein the properties-imparting compound comprises a gadolinium complex of DOTA (1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraacetic acid), DTPA (diethylenetriaminopentaacetic acid), DO3A (1,4,7,10-tetraazacyclododecan-1,4,7-triacetic acid), HPDO3A (10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecan-1,4,7-triacetic acid), TRITA (1,4,7,10-Tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclotridecane), TETA (1,4,8,11-Tetrakis(carboxymethyl)-1,4,8,11-Tetraazacyclotetradecane), BOPTA (4-carboxy-5,8,11-tris(carboxymethyl)-1-phenyl-2-oxa-5,8,11-triazatridecan-13-oic acid), NOTA (1,4,7-triazacyclononane-N,N′,N44-triacetic acid), PCTA (3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid), DOTMA ((alpha, alpha′, alpha″, alpha′″)-tetramethyl-1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraacetic acid), AAZTA (6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid) and HOPO (1-hydroxypyridin-2-one).

11. The method of claim 9, wherein the properties-imparting compound is: an iodinated compound of formula (III): ##STR00064## with X.sub.1 to X.sub.4 and L as defined in claim 1 and m representing 1, 2, 3 or 4, or a polymer comprising an iodinated moiety of formula ##STR00065## with m representing 1, 2, 3 or 4.

12. The method of claim 9, wherein the properties-imparting compound is: a rhodamine derivative of formula (Va): ##STR00066## with X.sub.1 to X.sub.4 and L as defined in claim 1, R.sup.1, R.sup.2, R.sup.3 and R.sup.4 each independently selected from a hydrogen atom or a C.sub.1-C.sub.6 alkyl group, and R.sup.5 selected from a hydrogen atom, a COOH or a C(O)OC.sub.1-C.sub.6 alkyl group; a cyanin derivative of formula (Vb): ##STR00067## with X.sub.1 to X.sub.4 and L as defined in claim 1, p′ being 0 or 1, q′ being 0 or 1 if p′ is 0 then R.sub.k is H, if q′ is 0 then R.sub.j is H, if p′ is 1 and q′ is 1, then R.sub.j and R.sub.k are both H or taken together, form a-CH.sub.2CH.sub.2CH.sub.2— bridging group, R.sub.d is selected from H and SO.sub.3Na, and R.sub.e is H or taken together, R.sub.d and R.sub.e form a-CH.sub.2CH.sub.2CH.sub.2- or CHCHCH— bridging group, R.sub.g is selected from H and SO.sub.3Na, and R.sub.f is H, or taken together, R.sub.f and R.sub.g form a-CH.sub.2CH.sub.2CH.sub.2- or CHCHCH— bridging group, R.sub.h being selected from a (C.sub.1-C.sub.6)alkyl group, optionally substituted with a SO.sub.3- or a COOH group, R.sub.i being selected from a (C.sub.1-C.sub.6)alkyl group, optionally substituted with a SO.sub.3Na or a COOH group, or a fluorescein derivative of formula (Vc): ##STR00068## with X.sub.1 to X.sub.4 and L as defined in claim 1, and R′ representing H, or a-CH.sub.2CH.sub.2COOH or —CH═CHCOOH group.

13. The method of claim 2, wherein the properties-imparting compound is a hydrophilic block copolymer with a block comprising repeated units A or a gradient-copolymer with a block a region in comprising repeated units A, said repeated units A comprising the photoactive aryl-azide moiety of formula (I) as defined in claim 1 with the nitrogen atom covalently bound to the surface of the surface-modified polymeric substrate, said hydrophilic block- or gradient-copolymer imparting anti-fouling properties.

14. The method of claim 3, wherein the properties-imparting compound is a hydrophilic block copolymer with a block comprising repeated units A or a gradient-copolymer with a block a region in comprising repeated units A, said repeated units A comprising the photoactive aryl-azide moiety of formula (I) as defined in claim 1 with the nitrogen atom covalently bound to the surface of the surface-modified polymeric substrate, said hydrophilic block- or gradient-copolymer imparting anti-fouling properties.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Scheme depicting the grafting method of the invention, using clip chemistry. The stars attached to the aryl-azide and aryl-amino moieties represent the properties-imparting compounds of the invention before and after grafting.

(2) FIG. 2. A,B: Possible ways of attachment of Gd-DTPA-biN3 after UV-irradiation on various polymer surfaces. C: Visibility of PP-mesh via MRI after UV treatment with Gd-DTPA-biN3.

(3) FIG. 3. A: Scheme of Gd-DOTA-N3 attachment on various polymer surfaces. B: Typical PP-mesh used for functionalization. C: Detection of PP-mesh via MRI after UV-treatment with Gd-DOTA-N3 D: Visibility of Gd-DOTA-N3 functionalized PP-mesh in vivo in rats. F: Position of untreated and treated PP-mesh in rat. G: Image of implantation side of PP-mesh in rat.

(4) FIG. 4. A: Scheme of clip-modified Rhodamine B attached to polymer surface after UV irradiation. B,C: Fluorescence images of PLA treated with clip-modified Rhodamine B (B) and untreated PLA (C). D:PLA surface visualized with optical microscopy.

(5) FIG. 5. A: Scheme of Gd-DTPA attached to various polymer surfaces via a clip-modified polysarcosine spacer. B: Visualization of PCL foil via MRI modified with 4-Azidoaniline initiated polysarcosine, ω-Gd-DTPA terminated.

(6) FIG. 6. Left: Scheme of polysarcosine covalently linked to polymer surface via the clip moiety. Right: Anti-adherence properties of PLA and PP surfaces untreated (control) and modified with polysarcosine.

(7) FIG. 7. A: Reduction of biofilm formation on PP and PLA surfaces after UV-functionalization with clip containing poly(2-oxazoline). B: Decrease in contact angle of PLA surface after treatment with poly(2-oxazoline). C: Schematic representation of attachment of (2-methyl-2-oxazoline)-b-poly(2-(4-azidophenyl)-oxazoline) block copolymer on polymer substrate.

(8) FIG. 8. Top: Scheme of Rhodamine B attached to various polymer surfaces via a clip-containing poly(2-oxazoline) copolymer spacer. Bottom: Fluorescence images of various polymer surfaces modified with the poly(2-oxazoline) copolymer before and after 15 h at 100° C. in H.sub.2O.

(9) FIG. 9. A: schematic representation of the substrate-modified films. B: MRI imaging of various polyester substrates (from left to right: PCL, PLA and PLGA) grafted with a DTPA aryl-amide moiety (7T, spin echo sequence with inversion, T1=1300 ms). C. MRI imaging of various polyester substrates grafted with a DTPA aryl-amide moiety (7T, spin echo sequence with inversion, T1=1300 ms), at t=0 and after 2 months incubation in a PBS medium (pH=7.4) at 37° C.

(10) FIG. 10. A: schematic representation of the substrate-modified films. B: biocompatibility assessment of substrates grafted with a DOTA aryl-amide moiety with a cytotoxicity assay by direct contact (left) and with a cell proliferation assay (right) on L929 mouse fibroblasts (n=5). C. Histological evaluation of the inflammatory response of substrates grafted with a DOTA aryl-amide moiety. Histological section (HES, ×5) with muscle, muscle tissues, fat tissues, mesh, inflammation, fibrosis (top row) and grading of inflammation in implanted rats (n=4) (bottom row).

(11) FIG. 11. XPS full spectra of untreated and POx modified polymeric surfaces. Peak assignment is shown exemplarily for the PP samples. Spectra of POx modified surfaces have been offset by 30×10.sup.3 counts/s along the y-axis for better visibility.

(12) FIG. 12: Biofilm formation of S. epidermidis stained with crystal violet on untreated (i.e. not modified) PS (top rows) and on Pox treated (i.e. modified with the polymers described in example 6) PS (bottom rows) with (A) a random copolymer and (B) a gradient copolymer.

EXAMPLES

(13) The present invention will be illustrated through the following examples, which are not to be construed as limiting the scope of the invention in any way.

(14) 1. Functionalization with 4-Azidoaniline Modified Gd-Diethylene Triamine Pentaacetic Acid (Gd-DTPA-biN.sub.3) Complex

(15) Clip-Synthesis:

(16) Under argon-atmosphere 2.2 eq 4-Azidoaniline hydrochloride were dissolved in anhydrous DMF and 2.2 eq TEA. 1 eq diethylene triamine pentaacetic dianhydride was added to the clear solution and the reaction mixture heated to 50° C. for 2 h and left stirring at RT over night in the dark and under Ar-atmosphere. The solvent was removed under high vacuum and the residue suspended in MeOH/EtOH and precipitated in cold diethylether/chloroform (50/50; v/v). Precipitation was repeated two more times, followed by dissolution in H.sub.2O and lyophilization to obtain DTPA-biN.sub.3 as a yellowish powder.

(17) 1 eq of DTPA-biN.sub.3 and 10 eq pyridine were dissolved in H.sub.2O and shaken for 30 min at 40° C. 2 eq GdCl3.6H.sub.2O were added and the reaction mixture shaken over night at 40° C. The precipitated product was dissolved in an access of H.sub.2O and treated with Chelex 100 to remove free Gd. The treatment was repeated until no further free Gd was detected with the MTB-test.

(18) ##STR00050##
Surface-Functionalization:

(19) Clean polymer (e.g. PLA, PLA-Pluronic-PLA, PLGA,PCL, PP) surface (film, mesh, pressed pellet) was covered with 1-5 g/L clip in degassed MeOH and irradiated for 5-30 min at 254 nm. Subsequently the surface was rinsed with H.sub.2O and EtOH.

(20) MRI Imaging

(21) Material.

(22) Bruker 7T BIOSPEC 70/20, “mini-imaging” configuration (gradient BGA12 675 mt/m, resonator “bird cage” 35 mm). After a set of marker gradient echo sequences, a spin3D echo sequence was acquired (FOV 3*3*1 cm matrix 128*128*48, TR=3000 ms, TE=8 ms (TEeff=16), RF=8, acquisition time of 0:51) with an inversion delay of 1300 ms.

(23) MGE 3D sequences with TR/TE 110/3 ms and angles of 75, 30 and 15° were acquired.

(24) Assumptions as to the binding of the nitrene to the polymeric substrate are presented in FIGS. 2 and 9A, as well as the visibility by MRI measurements (FIGS. 2C and 9B).

(25) MRI imaging on PCL, PLA and PLGA films (substrates), the surface of which has been grafted with the DTPA-N3 described above is described on FIG. 9B. These images were obtained after submitting the surface-modified substrates to a spin echo sequence with inversion (7T, T1=1300 ms).

(26) Stability of MRI-visibility for PP modified meshes after 2 months in saline phosphate buffer (pH 7.4, 37° C.) is shown in FIG. 9C. Surface-modified substrates with a thread-like shape were also successfully tested for stability: the image obtained after two months in a PBS medium (pH=7.4) at 37° C. does not show any image degradation (see FIG. 9C).

(27) 2. Functionalization with 4-Azidoaniline Modified Gd-2-(4,7,10-triacetic acid)-1,4,7,10-tetraazacyclododecan-1-yl)pentanedioic Acid, (Gd-DOTA-N.sub.3) Complex

(28) Clip-Synthesis:

(29) In an evacuated schlenk-flask 1 eq 4-azidoaniline hydrochloride were dissolved in anhydrous DMF. 1.2 eq TEA and 1.2 eq DOTA-GA anhydride (2,2′,2″-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid) were added. The reaction mixture was stirred for 3 h at 45° C. and continued to be stirred at RT over night. The solvent was removed under high vacuum and mild heating. The residue was dissolved in MeOH/CHCl.sub.3 (2/1; v/v) and precipitated in diethyl ether. Subsequently the precipitate was centrifuged, dried, dissolved in H.sub.2O and lyophilized to obtain DOTA-N.sub.3 as a yellow-brownish powder.

(30) 1 eq of DOTA-N.sub.3 and 10 eq pyridine (or NaOH) were dissolved in H.sub.2O (pH˜6.5). 2 eq GdCl3.6H.sub.2O were added and the reaction mixture shaken for 2 h to 15 h at 40-60° C. The solution was diluted with more H.sub.2O and treated with Chelex 100 to remove free Gd. The treatment was repeated until no further free Gd was detected with the MTB-test.

(31) ##STR00051##
Surface-Functionalization:

(32) Clean polymer (e.g. PLA, PLA-Pluronic-PLA, PLGA,PCL, PP, PEEK, PU) surfaces (film, mesh, pellet) heated to temperatures from RT to 80° C. were covered with 1-20 g/L clip in degassed MeOH (e.g. by spraying), air dried and irradiated for 5-10 min at 254 nm. Subsequently the surface was rinsed with H.sub.2O and EtOH. The irradiation step was optionally repeated up to 5 time to increase surface coverage. Final purification in H.sub.2O for 20 min in ultrasonic bath.

(33) Implantation in Rats and Imaging

(34) Control (untreated) and surface-modified surgical meshes (1 cm×2 cm) are desinfected using 70% ethanol and are sterilized by UV irradiation.

(35) 4 female rats are implanted with one control and one surgical mesh (2 meshes per rat). The prostheses are implanted in the dorsal muscle lodges. The control mesh is implanted in the left lodge, while the surface-modified mesh is implanted in the right lodge.

(36) MRI of the implanted rats are then carried out, using Spin Echo and Gradient Echo sequences at 9T, acquiring data for 30-40 minutes. The experiments were carried out on the BioNanoNMRI small animal MRI platform of the Universite de Montpellier.

(37) A scheme depicting the structure of the surface-modified substrate thus obtained is presented in FIG. 3 (A). FIG. 3 also shows a surgical mesh whose surface has been modified as above (B), and its visibility in MRI (B). The surface-modified mesh of the invention was implanted in a mouse (G), and the visibility of the implanted surgical mesh of the invention in vivo by MRI (D, F).

(38) Biocompatibility of Modified Surfaces (MRI)

(39) a) Cytotoxicity Assay Via Direct Contact Method

(40) L929 cells (Sigma-Aldrich) were seeded at 1.7.Math.10.sup.4 cells per well in a 24-well plate and allowed to attach overnight under appropriate atmosphere. Polymers PLA and PP with and without Gadolinium complex were cut in order to cover about 1/10 of the well surface (as mentioned in ISO 10993-5). Decontamination was realized: first step with ethanol 70% followed by 3 washing step with PBS-penicillin/streptomycin 10% and then PBS only.

(41) The cell growth medium was replaced and the decontaminated polymer films were placed on the top. After 48 h incubation at 37° C. 5% CO.sub.2, polymers were removed and cell viability was assessed by using Prestoblue® cell viability assay (Invitrogen, A13261) according to manufacturer's instructions. Briefly, Prestoblue® was added at 10% in growth medium and the fluorescence at 590 nm was measured after 45 minutes incubation. Wells without addition of polymers films are used as controls (n=4).

(42) b) Cell Proliferation Assay

(43) Polymers PLA and PP with and without Gadolinium complex were cut in order to cover 24-well plate surfaces (1.9 cm.sup.2). Polymer discs were swabbed with paper soaked with ethanol 70% and then rinse with 3 baths of PBS-penicillin/streptomycin 10% and then with PBS only.

(44) L929 cells (Sigma-Aldrich) were seeded on top of polymer (in the center) held by ring, at 2.10.sup.5 cells per well in a 24-well non-treated plate and incubated under appropriate atmosphere for about 2 h, time for cells to adhere. Then after washing non-adherent cells with PBS, fresh growth medium was added. Cell proliferation at 24 h, 48 h and 120 h was assessed by using Prestoblue® cell viability assay (Invitrogen, A13261) according to manufacturer's instructions. Wells without addition of polymers films are used as controls (n=5).

(45) The above in vitro biocompatibility tests, the results of which are summarized in FIG. 10B, demonstrate that the method of the invention leads to non-toxic materials.

(46) Histological Evaluation of the Inflammatory Response

(47) Meshes were implanted into back muscle tissue of 4 rats. Each rat received modified (Gadolinium complex) and no modified meshes on the other side. Implant specimens were recovered after 1 month implantation and were fixed with 10% formalin, paraffin-embedded, sectioned, and stained with hematoxylin-eosin-safran HES (RHEM platform, Montpellier). Lesion intensity (i) and spreading (d) were graded by experienced pathologist of RHEM technical facilities. Lesion intensity graded from i0 (no lesion) to i4 (severe) according to the presence of inflammatory zones. Lesion spreading graded from d0 (no lesion) to d2 (peripheral distribution). Results were compared with one control rat (without meshes implantation).

(48) This in vivo biocompatibility test, the results of which are summarized in FIG. 10C, demonstrate that the inflammation observed with modified meshes (results depicted by the plain squares) is due to surgery rather than to the surface modification of the material: the intensity of the inflammatory response as well as its spreading are less than or equivalent to those observed with unmodified (i.e. pristine) meshes (see results represented by the dotted squares).

(49) 3. Functionalization with 4-Azidoaniline Modified Rhodamine B

(50) Clip-Synthesis:

(51) 1.2 eq Rhodamine B isothiocyanate were dissolved in anhydrous DMF. 2 eq DIPEA and 1 eq 4-azidoaniline hydrochloride were added and the reaction mixture was shaken for at least 3 d at 40° C. The crude product was purified via Sephadex LH20 column (MeOH as eluent).

(52) ##STR00052##

(53) Surface-Functionalization:

(54) Clean polymer (e.g. PLA, PP) surface (e.g. film, pressed pellet) was covered with 1-20 g/L clip in degassed MeOH and irradiated for 1-20 min at 254 nm. Subsequently the surfaces were rinsed with H.sub.2O and EtOH.

(55) Fluorescence Measurements

(56) Material.

(57) Leica fluorescence microscope, 20× magnified, green excitation wavelength (555 nm). A scheme depicting the structure of a surface-modified PLA substrate thus obtained is presented in FIG. 4 (A). FIG. 4 also shows fluorescence images and optical microscopy of the fluorescent surface-modified PLA substrate.

(58) 4. Functionalization with 4-Azidoaniline Initiated Polysarcosine, ω-Gd-DTPA Terminated

(59) Clip-Synthesis:

(60) Sarcosine-N-Carboxyanhydride.

(61) First, 1 eq sarcosine were freshly grounded and dried for 1.5 h under high vacuum. Subsequently the powder was dissolved in anhydrous THF, 1.3 eq limonene, 2 eq diphosgene were added slowly under a steady flow of argon. The reaction mixture was heated to 65° C. for 2 h. After cooling to room temperature the reaction mixture was flushed for another 3 h with argon into two gas washing bottles filled with aqueous 20% NaOH solution. Next, the solvent was removed under vacuum until a yellow solid remained, which was then dissolved in dry THF. After addition of 20 mL petrolether the suspension was vigorously stirred for 30 min while cooled with an ice-bath. The precipitate was allowed to settle for 4 h under continued cooling. The clear supernatant was removed carefully with a syringe under argon atmosphere. The procedure was repeated one more time using petrolether and allowing precipitation over night in the freezer (−20° C.). After drying the solid under high vacuum, the raw product was sublimated (95° C., <10.sup.−2 mbar) under argon atmosphere, which yielded a white powder of sarcosine-NCA.

(62) ##STR00053##

(63) 4-azidoaniline initiated polysarcosine. Initiator stock solution was prepared with 4-azido-aniline hydrochloride, TEA and dry benzonitrile. Appropriate amounts of the initiator stock solutions were added to sarcosine-NCA dissolved in dry benzonitrile (c.sup.˜10 mg/mL). The reaction mixture was stirred at room temperature between 12 h and 7 d depending on the degree of polymerization. The polymer was precipitated twice in cold diethyl ether (10-20 fold of volume of polymer solution). After removal of the solvent and drying, the polymer was redissolved in H.sub.2O and lyophilized. White to light yellow powders were obtained.

(64) ##STR00054##

(65) 4-Azidoaniline Initiated Polysarcosine, ω-Gd-DTPA Terminated.

(66) 1 eq polysarcosine were dissolved in anhydrous DMAc. 2 eq of an acitivated form of DTPA (e.g. anhydride, thiol ester) were added (and 5 eq AgOTf together with the thiol ester) and stirred over night at RT. After removal of the solvent the residue was dissolved in CHCl.sub.3 and precipitated in cold diethyl ether. Final purification was achieved via Sephadex LH20 column (MeOH as eluent).

(67) 1 eq of polymer and 10 eq pyridine were dissolved in H.sub.2O and shaken for 2 h at 40° C. 2 eq GdCl3.6H.sub.2O were added and the reaction mixture shaken over night at 40° C. The precipitated product was dissolved in an access of H.sub.2O and treated with Chelex 100 to remove free Gd. The treatment was repeated until no further free Gd was detected with the MTB-test.

(68) ##STR00055##

(69) Surface-Functionalization:

(70) Clean polymer (e.g. PLA, PCL, PP, PLGA) surface (e.g. film, mesh) was covered with 1-20 g/L polymer-clip in degassed MeOH and irradiated for 5-10 min at 254 nm. Subsequently the surfaces were rinsed with H.sub.2O and EtOH.

(71) Material.

(72) Bruker 7T BIOSPEC 70/20, “mini-imaging” configuration (gradient BGA12 675 mt/m, resonator “bird cage” 35 mm). After a set of marker gradient echo sequences, a spin3D echo sequence was acquired (FOV 3*3*1 cm matrix 128*128*48, TR=3000 ms, TE=8 ms (TEeff=16), RF=8, acquisition time of 0:51) with an inversion delay of 1300 ms.

(73) MGE 3D sequences with TR/TE 110/3 ms and angles of 75, 30 and 15° were acquired.

(74) A scheme depicting the structure of a surface-modified substrate thus obtained is presented in FIG. 5 (A). FIG. 5 also shows the MRI image of such a surface-modified PCL substrate of the invention.

(75) 5. Functionalization with 4-Azidoaniline Initiated Polysarcosine

(76) Clip-Synthesis:

(77) Sarcosine-N-Carboxyanhydride. Described in Chapter 4.

(78) 4-Azidoaniline Initiated Polysarcosine. Described in Chapter 4.

(79) Surface-Functionalization:

(80) The polymers were dissolved in degassed methanol yielding concentrations between 0.1 to 50 g/L (in general the higher the degree of polymerization the higher the concentration has to be). Polylactic acid (PLA) and polypropylene (PP) surfaces were washed prior modification for 15-30 min in methanol in ultrasonic bath. After drying for another 15 min in high vacuum, the surfaces were heated to 60° C. On the warm surfaces the polymer solution was sprayed using an airbrush. Subsequently the surfaces were irradiated for 1-30 min at 254 nm. After irradiation the surfaces were washed in methanol or ethanol for 5-10 min. The procedure was repeated up to 5 times to improve the final result. Finally the surfaces were washed with ethanol in ultrasonic bath (unless substrate not stable in ultrasonic bath, then surface was just rinsed thoroughly) and dried under vacuum.

(81) Antifouling Effect

(82) S. epidermidis ATCC49461 and E. coli CFT073 strains were used for these experiments.

(83) The bacterial adhesion study was carried out using a technique adapted from Balasz et al (Biomaterials 25 (11) (2004) 2139-2151). The plates are immersed in wells containing the bacterial strain with an OD.sub.600 (optical density at 600 nm) of 0.05, diluted in culture medium. After 1 h, the plates are removed from the wells, vigorously rinsed 3 times with sterile water, and then immersed in a neutral medium (AP or PBS). At 24 hours, the plates-adhering bacteria are recovered after vortexing and sonication in sterile saline. The bacteria were quantified by serial dilutions and plating on Mueller Hinton agar culture media. The most adherent bacteria are detached by transferring each face of the plates fifteen times on Mueller Hinton agar media. The bacteria counting is conducted after an overnight incubation at 37° C. The total adherent bacteria population is obtained by adding all cultured bacteria. The results are expressed as CFU (colony forming units). A verification of the bacteria identity is made using MALDI-TOF mass spectroscopy (Vitek-MS, BioMerieux).

(84) A scheme depicting the structure of a surface-modified substrate thus obtained is presented in FIG. 6 (A). FIG. 6 also shows the anti-adherence performance of such surface-modified PLA and PP substrates of the invention, with regard to S. epidermis.

(85) 6. Functionalization with poly(2-methyl-2-oxazoline)-co-poly(2-(4-azidophenyl)-oxazoline) Copolymers

(86) Clip-Synthesis:

(87) 2-(4-Azidophenyl)-oxazoline. 1 eq 4-azidobenzoic acid was dried for 1 h under high vacuum. Subsequently 5 eq thionyl chloride and dry THF were added. The reaction mixture was stirred for 3 h at 70° C. under argon atmosphere. The solvent was removed under reduced pressure and the crude product crystallized in cyclohexane. After removal of the supernatant and drying 4-azido-benzoyl chloride as beige crystals were obtained.

(88) ##STR00056##

(89) Next, 1 eq 4-azido-benzoyl chloride were dissolved in chloroform. 1.1 eq 2-bromoethylamine hydrobromide and potassium hydroxide were dissolved in H.sub.2O and cooled with an ice-bath. To the cold aqueous solution the organic solution was added and stirred for 15 min under continued cooling and for another 15 min at room temperature. The phases were separated and the organic phase washed with H.sub.2O, dried with MgSO.sub.4 and filtered. The solvent was removed yielding N-(2-bromoethylamine)-4-azidobenzamide as a light yellow solid.

(90) ##STR00057##

(91) Finally, 1 eq N-(2-bromoethylamine)-4-azidobenzamide and 1.1 eq potassium hydroxide were dissolved in anhydrous methanol and stirred over night at room temperature under argon atmosphere. After removal of the solvent the residue was dissolved in dichloromethane and washed 3× with H.sub.2O. Subsequently the organic phase was treated with MgSO.sub.4, filtered and dried. 2-(4-azidophenyl)-oxazoline was obtained.

(92) ##STR00058##
Poly(2-oxazoline).

(93) For block-copolymers, an adequate amount of 2-(4-azidophenyl)-oxazoline (n eq) was added to an evacuated flask and dried further under high vacuum. The initiator methyl triflate (MeOTf, 1 eq), and dry acetonitrile (ACN, final monomer concentration <3 M) were also added under inert conditions. For gradient copolymers, the first block of 2-(4-azidophenyl)-oxazoline was copolymerized with a small amount 2-methyl-2-oxazoline (MeOx) to spread the functional groups within the polymer. The reaction mixture was then stirred for 3-7 d (depending on the block length) at 80° C. Next MeOx was added under argon flow to the reaction mixture. Depending on the set degree of polymerization the polymerization was carried out for another 1-7 d at 80° C. The reaction was terminated with 3 eq 1-BOC-piperazine, which was stirred for 5 h at 40° C. Subsequently an excess of potassium carbonate was added and the mixture stirred over night at room temperature. After centrifugation and filtration, the solvent was removed and the residue dissolved in a mixture of chloroform and methanol (1/2, v/v) followed by precipitation in cold diethyl ether (10-20 fold of volume of polymer solution). After a second precipitation the polymer was dried, dissolved in H.sub.2O and lyophilized. White to dark yellow powders were obtained.

(94) ##STR00059##

(95) Random copolymers are obtained through a similar procedure wherein the monomers are all present during polymerization.

(96) Surface-Functionalization:

(97) As described in chapter 5. Results are shown in FIG. 11.

(98) Antifouling Effect: Biofilm Formation

(99) S. epidermidis ATCC49461 and E. coli CFT073 strains were used for these experiments.

(100) Quantification Using Crystal Violet:

(101) The plates are immersed in wells containing the bacterial strain with an OD.sub.600=0.05, diluted in culture medium. After 72 h incubation at 37° C., the plates are removed from the wells, and vigorously rinsed 3 times with sterile water.

(102) The plates are then placed for 10 minutes in 0.1% crystal violet to stain the bacteria involved in the biofilm. They are then washed 3 times with sterile water to remove excess dye. The bacteria are then precipitated with 250 μl of DMSO. The resulting solution was assayed using a spectrophotometer to measure the OD.sub.600.

(103) A scheme depicting the structure of a surface-modified substrate thus obtained is presented in FIG. 7 (C). FIG. 7 (A) shows the reduction of biofilm formation on such a surface-modified PP and PLA substrate, as compared with a non-grafted control.

(104) Contact Angles

(105) Contact angles were measured via a progressive scan CCD camera (Dataphysics OCAH200) and analyzed using ImageJ software. FIG. 7(B) shows decrease in contact angle of such a surface-modified PLA substrate (B), as compared with an untreated (non-grafted) PLA substrate.

(106) Crystal Violet Assay for Quantification of Submerged Bacterial Biofilms

(107) Material:

(108) Polymer-coated 6-well plate Tryptic Soy Broth (TSB, Becton-Dickinson) 0.1% Crystal Violet (Carl Roth) in H.sub.2O 1×PBS H.sub.2O 33% acetic acid (Carl Roth) Photometer and single use cuvettes
Strain: Staphylococcus epidermidis RP62a
Setting Bacterial Biofilms.

(109) The bacteria were grown overnight in 2 ml TSB in 13 ml culture tubes at 37° C. shaking with 220 rpm. The absorption of the culture was measured at 600 nm (OD.sub.600 nm) and a preculture of 20 ml (in 100 ml flasks) was started with an OD.sub.600 nm=0.05 in TSB. The preculture was incubated for 4-6 h at 37° C. shaking with 220 rpm to have a culture in the exponential growth phase. The OD.sub.600 nm was measured and the culture was diluted in TSB to an OD.sub.600 nm=0.05. Each well was filled with 4 ml of the bacterial solution. The plate was incubated at 37° C. for 24 h to allow formation of the submerged biofilm.

(110) Staining and Quantification of Biofilms.

(111) The multi-well plate was flipped over to discard the medium. The wells were then gently washed three times with PBS to remove unbound cells. The biofilms were fixed by heating the plate (without the lid) to 65° C. for 30 min. To stain the biofilms, 1 ml 0.1% crystal violet was added to the wells and incubated for 3 min. The dye was discarded and the wells were washed with H.sub.2O until no more dye was found in the water (at least 3 times). Results. The obtained results are shown on FIG. 12. It appears that gradient copolymers prevent more efficiently the formation of the biofilm.

(112) 7. Functionalization with poly(2-methyl-2-oxazoline)-co-poly(2-(4-azidophenyl)-oxazoline) Copolymers, Rhodamine B Terminated

(113) Clip-Synthesis:

(114) 2-(4-Azidophenyl)-oxazoline. As Described in Chapter 6.

(115) Poly(2-oxazoline). As Described in Chapter 6.

(116) Poly(2-oxazoline), Rhodamine B Terminated.

(117) 1.2 eq Rhodamine B isothiocyanate were dissolved in anhydrous DMF. 1 eq DIPEA and 1 eq poly(2-oxazoline) were added and the reaction mixture was shaken for at least 3 d at 40° C. The crude product was purified via Sephadex LH20 column (MeOH as eluent).

(118) Surface-Functionalization: As Described in Chapter 5.

(119) Fluorescence Measurements

(120) Material.

(121) Leica fluorescence microscope, 20× magnified, green excitation wavelength (555 nm).

(122) A scheme depicting the assumed structure of a surface-modified substrate thus obtained is presented in FIG. 8 (A). FIG. 8 also shows fluorescence images of various surface-modified substrates thus obtained, before and after being heated at 100° C. in H.sub.2O for 15 h. These results demonstrate the stability of the surface-modified substrates of the invention.