Vancomycin Functionalized Nanoparticles for Bactericidal Biomaterial

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Vancomycin functionalized nanoparticles for bactericidal biomaterial surfaces Loic Pichavant, Hélène Carrié, Minh Ngoc Nguyen, Laurent Plawinski, Marie-Christine Durrieu, and Valérie Héroguez Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01727 • Publication Date (Web): 03 Mar 2016 Downloaded from http://pubs.acs.org on March 8, 2016

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Vancomycin functionalized nanoparticles for bactericidal biomaterial surfaces Loïc Pichavant a,b, Hélène Carrié a,b, Minh Ngoc Nguyen a,c, Laurent Plawinski b, MarieChristine Durrieu b and Valérie Héroguez a*

a

CNRS UMR5629, Laboratoire de Chimie des Polymères Organiques, IPB-ENSCBP, Université

de Bordeaux, 16 avenue Pey Berland, F-33607 Pessac, France

b

CNRS UMR5248, Institut de Chimie & Biologie des Membranes & des Nanoobjets, Université

de Bordeaux, INP Bordeaux, 2 rue Robert Escarpit F-33607 Pessac cedex France

c

Faculty of Chemistry, VNU University of Science, Hanoi, 19 Le Thanh Tong, Hoan Kiem,

Hanoi, Vietnam

KEYWORDS: ROMP; dispersion polymerization; functionalized nanoparticles; antibacterial biomaterial.

1 Environment ACS Paragon Plus

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ABSTRACT

In this paper, we describe a simple and powerful way to synthesize antibacterial biomaterials with applications as implants in orthopaedic surgery. Such implants are obtained by covalently grafting onto the Ti90A16V4 alloy surface with Vancomycin-functionalized nanoparticles. Nanoparticles were produced by Ring-Opening Metathesis Polymerization of α-norbornenyl-ωVancomycin poly(ethylene oxide) macromonomers. Vancomycin is an interesting candidate because of its use in the field of implant associate infection as it is a glycopeptide which acts on bacterial walls. As a consequence, Vancomycin does not need to be released for it to be active. In the first part of this paper, the synthesis and the complete characterization of these materials are described. In a second part, the in vitro antibacterial behavior is analyzed and discussed.

1. Introduction:

Bacterial infections are the most current complication which could occur during the fitting of implantable biomaterials1,2,3,4 It can be the cause of morbidity and mortality, and take place in 2% to 6% of the cases despite antibioprophylaxis. In this case, depending on the implantation site and the nature of the biomaterial, these rates could reach 40% of cases for ventricular assist device5,6 Bacteria responsible for this kind of infections are (in 50% of the cases) Staphylococcus epidermidis or Staphylococcus aureus, and mostly happens in per-operative conditions (nosocomial infections)7. Even if antibioprophylaxis considerably decreases the rate of per-


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operatory infection, it presents risks of allergy, side effects or bacterial resistance. Moreover, standard antibiotic protocols present little efficiency because of the immuno-incompetent zone around the implant, reduced sensitivities of bacteria growing in a biofilm and difficulties of the drug reaching the site of infection due to poor penetration of antibiotics into bone and joint spaces. It is for this reason why local antibiotic treatments are more developed and employed1,8,9. In recent papers, we presented a new biomaterial having applications as implant in orthopeadic surgery, and allowing the release of an antibiotic (Gentamicin Sulfate) only in the case of infection10,11. For that, antibiotic functionalized nanoparticles (NPs) were covalently grafted onto titanium surfaces. The active molecules were linked on NPs through a pH-sensitive bond (an imine bond). In case of infection, bacterial proliferation induces a local decrease of the pH, which leads to the hydrolysis of the imine bond and the deliverance of the drug. The synthesis of this kind of materials was perfectly described, and their efficiency was proved by in vitro tests. Another strategy to fight against bacterial infection is to develop a bactericidal biomaterial surface. For example, in 2009, K. Anseth et al. showed that polymerizable derivatives of Vancomycin might be useful to modify implant surface reducing adherent Staphylococcus epidermidis numbers by sevenfold when compared to the control surface12,13,14. No information was given about the drug density immobilized onto the biomaterial surfaces. Previously, Arimoto et al. have demonstrated that multi-valent polymers of Vancomycin containing 2 to 15-mers were capable of restoring antibacterial activity against Vancomycin resistant Enterococcus15,16,17. Vancomycin is a glycopeptide used in the prophylaxis and treatments of infections caused by Gram-positive bacteria such as Staphylococci. It acts by inhibiting the cell wall synthesis by binding to the C-terminal L-Lys-D-Ala-D-Ala motif present in cell wall precursors found in certain susceptible strains (Scheme 1)18,19. Contrary to Gentamicin Sulfate, which acts on the


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protein biosynthesis by its linkage on ribosome and consequently needs to be released and internalized, Vancomycin does not need to be released and can be perennially linked onto the biomaterial reducing bacterial resistance risks.

Vancomycin (Vanco-NH2)

L-Lys-D-Ala-D-Ala Scheme 1: Vancomycin structure and its complex formation with the L-Lys-D-Ala-D-Ala motif of the cell wall precursor18

Herein, we propose to synthesize a titanium bioactive material grafted with Vancomycinfunctionalized NPs (Scheme 2). Such NPs are obtained by Ring-Opening Metathesis coPolymerization (ROMP) of new α-norbornenyl-ω-Vancomycin poly(ethylene oxide) macromonomers with its ω-carboxylic acid functionalized homologue and norbornene (Nb) in dispersed medium. We previously demonstrated that ROMP of α-ω-polyethylene oxide macromonomers is a powerful route to prepare polynorbornene-based particles20. Our approach


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offers the advantage of flexibility in design and the functionality of macromonomers used for copolymerization can be adapted to produce particles with high tumor selectivity21,22 or pHresponsive drug delivery behavior23. Finally, the NPs were covalently linked onto amine functionalized titanium surfaces through an amide bond. The reason of our interest in NPs grafting is threefold: first, it allows an increase of the specific surface and also a higher Vancomycin density. Secondly, NPs can be view as a simply and flexible route to synthesize plurifunctional platform for elaboration of future innovating biomaterials functionalized with various drugs. Third, strengthening the Vancomycin concentration at a specific point could enhance antibacterial activity.

Vanco

Vanco Vanco Vanco

Vanco Vanco

Bacteria

Vanco

Vanco

Titanium biomaterial

Vanco

Titanium biomaterial

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Vanco Vanco Vanco

Scheme 2: Representation of the bioactive biomaterial

The first part of this paper describes the synthesis of these novel multi-valent Vancomycin NPs and their grafting onto Ti90A16V4 alloy. Thanks to surface observations made by scanning electron microscopy and a well-established quantification method, the density of Vancomycin linked onto the surfaces could be determined11,23. In the second part, the in vitro activity of the


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polymers has been evaluated through MIC (Minimum Inhibitory Concentration) and MBC (Minimum Bactericidal Concentration) tests in order to prove their antibacterial efficiency. Then immunofluorescent assays were performed onto the grafted materials to confirm the accessibility of Vancomycin.

2. Experimental part:

2.1. Materials:

Surgical-grade Ti90Al6V4 alloy samples (Ø = 5 mm; h = 3 mm; Ra = 5-6 µm) were purchased from Good Fellow, Belgium. Before use, samples were sonicated for 10 min in acetone, followed by a further 10 min sonication in ethanol. Anhydrous hexane (99%), ethanol (EtOH; 96%), dichloromethane (DCM; 99%), dicyclohexylcarbodiimide (DCC; 99%), N-hydroxysuccinimide (NHS; 98%), 3-aminopropyltriethoxysilane (APTES; 97%), triethylamine (TEA; reagent grade), bis(tricyclohexylphosphine) benzylidine ruthenium (IV) chloride (GC1, first generation Grubbs catalyst; stored in a glovebox filled with argon prior to use), norbornene (Nb; 99%) were purchased from Sigma Aldrich, France. Vancomycin hydrochloride hydrate was purchased from Alfa Aesar, France. N,N’-disuccinimidyl carbonate (DSC, 98%), 4-dimethylaminopyridine (DMAP, 99%) were obtained from Acros, France. All these chemical compounds were used as received. Dioxane (Scharlau), acetone (VWR) and N-N-dimethylformamide (DMF) were dried on molecular sieve 4 Å before used.


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2.2. Vancomycin functionalized NPs synthesis:

The first step of the formation of Vancomycin functionalized NPs was the synthesis of α-ωfunctionalized poly(ethylene oxide) macromonomers. α-norbornenyl-poly(ethylene oxide) (1) and α-norbornenyl-ω-carboxylic acid-poly(ethylene oxide) (3) were synthesized as described previously9. The α-norbornenyl-ω-Vancomycin functionalized macromonomer was synthesized in two steps. The macromonomers were characterized by 1H NMR using a 400 MHz Bruker spectrometer (CDCl3 or DMSO-d6 as solvent) and size exclusion chromatography (SEC) using a Varian apparatus equipped with TOSOHAAS TSK gel columns and a refractive index detector. THF and DMF were used as the solvent at a flow rate of 1 mL.min-1. Mass calibration was achieved with narrow dispersity poly(ethylene oxide) standards.

Synthesis of α-norbornenyl-ω-succinimidyl poly(ethylene oxide): α-norbornenyl poly(ethylene oxide) (1) (4.5 g, 1.5 mmol) was dissolved in 25 mL of anhydrous dioxane and DSC (2.3 g, 9 mmol), dispersed in 20 mL of anhydrous acetone, was added to the resulting solution. Then, DMAP (1.1 g, 9 mmol), dispersed in 15 mL of anhydrous acetone, was added slowly under magnetic stirring. The reaction was stirred at room temperature for 16 h. After this time, αnorbornenyl-ω-succinimidyl-poly(ethylene oxide) was directly precipitated in diethyl ether. Finally, several cycles of dissolution of the product in acetone and precipitation in diethyl ether were carried out in order to remove excess DSC and DMAP. The activated product was lyophilized in dioxane. Yield: 75%; Functionnalization: 95%. 1H NMR (CDCl3, 400 MHz): δ (ppm) Norbornenyl moiety, 5.85-6.04, 3.30, 3.11, 3.00, 2.84, 2.68-2.72, 2.25, 1.74, 1.62, 1.041.40, 0.41; EO moiety, 4.40, 3.40-3.80; Succinimidyl moiety, 2.77. (Supporting information)


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Synthesis of α-norbornenyl-ω-Vancomycin poly(ethylene oxide) (3): To a solution of Vancomycin (4.07 g, 1.08 mmol) in 30 mL of anhydrous DMF was added triethylamine (TEA, 3 mL, 46.84 mmol), a solution of α-norbornenyl-ω-succinimidyl poly(ethylene oxide) (3.12 g, 2.16 mmol) in 30 mL of anhydrous DMF and molecular sieves (4 Å). The reaction mixture was stirred at 38 °C for 18 h. After this time, this mixture was filtered through celite, precipitate in diethyl ether, filtrated and dried under vacuum. The product was purified by ultrafiltration using deionized H2O as solvent and regenerated cellulose membrane (5 kDa) to separate the product from unreacted Vancomycin and macromonomer. Finally, the product was lyophilized. Yield: 88%; Functionnalization higher than 99%.

1

H NMR (DMSO-d6, 400 MHz): δ (ppm)

Norbornenyl moiety, 5.85-6.04, 3.30, 3.11, 3.00, 2.84, 2.68-2.72, 2.25, 1.74, 1.62, 1.04-1.40, 0.41; EO moiety, 3.40-3.80; Vancomycin moiety, 6.44-7.66, 5.29-5.49, 4.06-4.47, 3.41-3.75 (overlapped by EO moiety peak), 2.73, 1.11-2.02, 0.84. (Supporting information)

Synthesis of Vancomycin-carboxylic acid functionalized NPs: Plurifunctionalized NPs were synthesized by Ring-Opening Metathesis coPolymerization (ROMP) of Nb with α-ωfunctionalized poly(ethylene oxide) macromonomers, following a scenario very similar to that described in a recent paper10. Solvents were degassed according to the freeze-pump-thaw procedure. The reaction was carried out at room temperature under inert atmosphere (glovebox). In a typical experiment, 30 mg (M= 823 g.mol-1; n= 3.6 × 10-5 mol) of Grubbs first generation complex were dissolved in 10 mL of DCM/EtOH solution (50/50% v/v). Both Nb (580 mg; M= 94 g.mol-1; n= 6.2 × 10-3 mol) and macromonomers (2) and (3) (m2 = 153 mg; Mn;2 = 4120 g.mol1

; m3 = 570 mg; Mn;3 = 4410 g.mol-1; n3 = 3n2 =1.1 × 10-4 mol) were first dissolved in 18 mL of

DCM/EtOH solution (35/65% v/v) (1 mL of this solution was used for the conversion


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measurements (t = 0)) and added to the catalyst solution. The mixture was stirred during 24 h. At the end of polymerization Ruthenium end-capped chains were deactivated by addition of 0.2 mL of ethyl vinyl ether. Macromonomer conversions were measured by SEC and Nb conversions were determined by gas chromatography (GC) with a VARIAN GC3900 using dodecane as internal standard. Then, the NPs were transferred to DMF to carry out the grafting step onto titanium surfaces: first DMF was added dropwise under stirring, and then dichloromethane and ethanol were evaporated under reduced pressure. NPs sizes in DCM/EtOH solution and in DMF were measured by Dynamic Light Scattering (DLS) using a MALVERN zetasizer Nano ZS equipped with He-Ne laser (4 mW; 633 nm). Before measurements, latexes were diluted about 800 times to minimize multiple scatterings caused by high concentration. The scattering angle used was 173°. For in vitro antibacterial tests, the NPs were also transferred into water. For that, first dichloromethane was evaporated, then water was added dropwise under stirring before ethanol was also evaporated. Finally, the NPs dispersion in water was purified by ultrafiltration.

2.3. NPs grafting onto titanium surfaces:

The NPs grafting was performed using a two-step process. First, titanium surfaces were functionalized with amine groups using APTES through a well-established protocol24: Briefly, titanium samples were outgassed at 150°C under vacuum (10-5 Torr) for 20 h. Then, silanization of the surface was performed by immersing the substrate in a solution of APTES (10-2 M) in anhydrous hexane under inert atmosphere (glovebox) for 2 h. Samples were washed three times in the glovebox under stirring, and then sonicated twice for 15 min (both steps have been


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performed using anhydrous hexane). Finally, samples were outgassed at 100°C under vacuum (10-5 Torr) for 4 h. Secondly, the NPs were covalently linked onto the titanium surface through the formation of an amide bond between the carboxylic acid groups of the NPs and the amine groups present on the surfaces (activated by NHS and DCC): Under inert atmosphere (glovebox), DCC (0.24 mg, 1.10 mmol; 73 eq.) and NHS (0.10 mg, 0.87 mmol; 58 eq.) were diluted in 2 mL of NPs dispersed in DMF (n-COOH = 1.5×10-5 mol). Finally, the mixture was deposited onto titanium materials and the resulting samples were stirred for 72 h at room temperature. The samples were then washed in three successive ethanol baths, dried and stored under inert atmosphere. The grafting step was carried out three times. Between two successive steps, the materials were rinsed in ethanol baths. The grafting density was characterized by SEM observations using a HITACHI S-2500 scanning electron microscope. For the NPs counting, pictures were carried out with a growth of 7000. This counting was achieved on six pictures obtained randomly on the titanium surfaces (three materials; two pictures per material). Surfaces were gold-coated before observations.

2.4. In vitro efficiency evaluation:

To evaluate biological changes in macromonomer and particle activity, we determined the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values for the new species. All MIC and MBC experiments were conducted at least in triplicate. Methicillin resistant Staphylococcus aureus BCB8 (MRSA BCB8; UPRES EA3826, Nantes) were cultured in Mueller-Hinton (MH) broth (Conda S.A.) at 37 °C for 15 h (overnight culture),


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without shaking. Bacterial culture was then diluted to 1 × 106 cfu.mL-1 using a 0.5 McFarland standard.

MIC and MBC experiments: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by the broth dilution method. From a 512 µg.mL-1 solution of Vancomycin, two-fold dilutions were performed in MH broth resulting in a range from 256 ̶ 0.0625 µg.mL-1. Then a bacterial suspension was added resulting in a final concentration from 170 ̶ 0.04 µg.mL-1 of Vancomycin and a bacterial suspension of 105 cfu. Cultures were incubated for 24 h at 37 °C. The MIC was defined as the lowest Vancomycin concentration at which no turbidity was observed. The MBC was determined by plating media from each of the above tubes after 24 h culture at 37 °C. First a 100-fold dilution was applied in physiological water and then 50 µL were deposited on trypticase soy (TS; Conda S.A) agar plates. As a control, we also used the drop plate method. For this, 5 µL of the same tubes used above were deposit on TS agar plates (Supporting information). The MBC was determined as the lowest Vancomycin concentration that results in a ≥ 99.9% decrease in the initial inoculum within 24 h. For MIC and MBC, data were presented as means ± standard derivation (SD), compared to each other using the Mann-Withney test and considered to be significantly different at p < 0.01.

Immunofluorescent Analysis of the Vancomycin functionalized titanium Surface: To determine the distribution and the accessibility of Vancomycin, the grafted surfaces were evaluated by indirect immunofluorescence using anti-Vancomycin antibody. Titanium samples were washed once with phosphate saline buffer (PBS; Euromedex, SouffelWeyersheim), once with PBS


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supplemented with 0.05 % (v/v) Tween 20 (T) and 5 % (wt/v) bovine serum albumin (blocking buffer) and then incubated with primary monoclonal anti-Vancomycin IgM from mouse (1:500, Abcam, Cambridge, UK) in blocking buffer, overnight at 4 °C. They were then washed two times with PBS-T, incubated in PBS-T for 30 min and treated with secondary AlexaFluor 488coupled anti-mouse IgG from donkey (1:500, Abcam, Cambridge, UK) in blocking buffer, for 2 h at room temperature. Finally, the samples were washed 3 times in PBS-T and incubated in PBS for 30 min. Stain was used in order to visualize samples by fluorescence microscopy (Leica DM5500 fluorescent microscope, Germany).

3. Results and discussion:

Our aim is to synthesize new bactericidal titanium surface for preventing bacterial infection. To synthesize these materials, first Vancomycin functionalized polymeric NPs were formed by ROMP in dispersion, then these NPs were covalently linked onto the titanium surfaces. Vancomycin has been chosen because it is currently one of the most efficient treatments for eradicating resistant Staphylococcus aureus. In addition, Vancomycin acts directly on the bacterial walls through the formation of a complex with the L-Lys-D-Ala-D-Ala motif of the cell, and it does not need to be internalized to be effective. That is why in this work, Vancomycin is perennially linked at the Titanium surface, limiting side effects and bacterial resistance.

In recent studies, Arimoto et al. described the synthesis of multivalent polymer of Vancomycin via the ROMP of a norbornene-modified Vancomycin monomer in a standard homogeneous system15,16,17. Nevertheless, they unsuccessfully tried to synthesize polymeric particles by ROMP


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in emulsion conditions (yield less than 4%). Herein, we present a versatile method allowing us to obtain NPs functionalized with Vancomycin and easily grafting onto a Titanium surface. By this way, we modify the surface of the biomaterial by locally introducing a high concentration of Vancomycin. We are also particularly interested in assessing how the spot distribution of Vancomycin can affect the bacterial proliferation.

3.1. Antibacterial biomaterial Synthesis:

The synthesis of such materials is divided into three parts. In the first part, α-ω-functionalized poly(ethylene oxide) macromonomers are synthesized. Secondly, these macromonomers are copolymerized with norbornene to form functionalized NPs. Finally, the synthesized NPs were covalently linked onto the titanium surfaces.

α-ω-functionalized poly(ethylene oxide) macromonomers: The synthesis of α-ω-functionalized macromonomers was divided into five steps. After each step, the macromonomers were characterized by 1H NMR and SEC in order to check their structures and their functionalization yields.


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Scheme 3: α-ω-functionalized poly(ethylene oxide) macromonomers

In the first step, α-norbornenyl-poly(ethylene oxide) macromonomer (1) was obtained by anionic ring-opening polymerization of ethylene oxide, which was initiated by 5-norbornene-2methanol that was deprotonated by DPMK. Then (1) was ω-functionalized with a carboxylic acid (Scheme 3): the alcohol function at the ω-end of the macromonomer, deprotonated by NaH, reacted with 2-bromoethyl acetate by nucleophilic substitution to form α-norbornenyl-ω-ester poly(ethylene oxide) macromonomer. Then the ester was hydrolyzed by NaOH to form the αnorbornenyl-ω-carboxylic acid poly(ethylene oxide) macromonomer (2). These three steps were described in a recent paper, and the structures and the functionalization yields of the macromonomers were check by 1H NMR and SEC10. (1) was obtained with a number-average molecular weight of about 3600 g.mol-1 calculated with the 1H NMR spectrum. The controlled nature of the anionic polymerization was confirmed by SEC (Table 1) (Ð = 1.09). All the chains of this macromonomer were functionalized with a norbornenyl entity. (2) was obtained with a number-average molecular weight of about 4120 g.mol-1, and a functionalization yield of about 75%. The number-average polymerization degree (DPn) of the two macromonomers (1) and (2),


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calculated with the 1H NMR spectrum, are comparable, which prove that the norbornenyl entity was not degraded during the ω-functionalization (Table 1). The ω-functionalization of the macromonomer with Vancomycin (Vanco) was divided into two steps (Scheme 4)25. First, an activated norbornenyl-poly(ethylene oxide) was formed by reacting (1) with N,N-disuccinimidyl carbonate in the presence of DMAP as catalyst and in anhydrous dioxane/acetone solvent mixture according to a recent publication26. Then, Vancomycin was linked via an amide bond by reacting its amine function with the activated macromonomer ester group in DMF and in the presence of triethylamine to form the αnorbornenyl-ω-Vancomycin poly(ethylene oxide) macromonomer (3). O

O O

N

O O

DMPA ; dioxane ; acetone

O n

O

N

O O

O O n

N O

n

(1)

O

O

O

OH

O

O

O

Vanco-NH2

N

O TEA ; DMF

O

(3)

O

H N Vanco

O n

O

Scheme 4: Synthesis of α-norbornenyl-ω-Vancomycinpoly(ethylene oxide) macromonomer

(3) was characterized by 1H NMR and SEC in order to check the structure and to determine the functionalization yield. The 1H NMR spectrum of (3) (Supporting information) illustrates the disappearance of the peaks corresponding to the succinimide entity (δ= 2.77 ppm) and the appearance of new characteristic peaks corresponding to the Vancomycin and more particularly a peak at δ= 0.84 ppm corresponding to six protons of the methyl groups of the drug27. The functionalization yield can also be calculated following equation 1, where Ivanco is the integration


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of the peak at δ= 2.77 ppm and INb is the integration of the peaks at 5.92 < δ< 6.13 ppm corresponding to the ethylenic protons of the norbornenyl entity.

=

/

equation 1

/

(3) was obtained with a quasi-quantitative functionalization yield after purification. The number-average polymerization degree calculated using the 1H NMR is similar to (1) which proves that the norbornenyl entity has not degraded during the ω-functionalization (Table 1). The number-average molecular weight calculated with this spectrum also corresponds to the expected weight. Nevertheless, the weight measured by SEC is higher than the theoretical one. This result can be explained by the fact that Vancomycin linked at the ω-end of the macromonomer involves a steric hindrance and an increase of the rigidity of the molecule, which can modify the hydrodynamic volume. Consequently, the apparent molecular weight can differ from the real molecular weight. The dispersity (Ð) remains low (1.20), reflecting a narrow distribution.

Table 1: Macromonomer characteristics determined by 1H NMR and SEC

; a Macrom.

; b

F (%) -1

(RMN)

(RMN) (g.mol )

;

Ð

(SEC)

/ ) (=

(g.mol-1)

(SEC)

(1)d

-

83

3640

3750

1.09

(2)d

75

93

4120

4440

1.20

(3)

> 99

87

5280

8240c

1.20c


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a

DPn = IPEO/2INb with IPEO = integration of the protons of the PEO linear chain, and INb = integration of the ethylenic protons of the norbornenyl group. b

Mn = 44DPn + MNb + Mω with MNb = molecular weight of the norbornenyl group and Mω = the molecular weight of the ω-function; 44 is the molecular weight of the ethylene oxide unit c

determined with DMF as solvent

d

previous results10

Functionalized nanoparticles: Functionalized NPs were obtained by Ring-Opening Metathesis coPolymerization of the α-ω-functionalized macromonomers with norbornene in dispersed medium initiated by the Grubbs I complex (Scheme 5). The formed polynorbornene, not soluble in the reaction medium (DCM/EtOH 35/65), precipitates during the reaction. The poly(ethylene oxide), soluble in the solvent, stabilizes the polynorbornene to form the core-shell NPs. By this way, the obtained NPs have an hydrophobic polynorbornene core and hydrophilic poly(ethylene oxide) shell28,29.

Scheme 5: Synthesis of functionalized NPs by ROMP in dispersed medium

Two kinds of NPs were synthesized: NP1 functionalized only with carboxylic groups (synthesized using macromonomers (1) and (2); [(1)] = 3[(2)]) for control, and NP2 functionalized with carboxylic acid groups and with Vancomycin (synthesized using


Biomacromolecules

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macromonomers (2) and (3); [(3)] = 3[(2)]). For each synthesis, Nb and macromonomers conversions were measured by GC and SEC respectively, using dodecane as an internal standard. In each case, the Nb conversions were higher than 99% and the macromonomer conversions were higher than 90%. By this way, we could estimate the linked Vancomycin concentration at about Cvanco = 128 mg per gram of latex by following the equation 2.

!

=

"×$ ×%& ' ("('* ('$ )

equation 2

With: - n3 the initial amount of monomer (3) - Mvanco the molecular weight of Vancomycin - mi the initial weight of compound i - π the conversion of the macromonomers For this calculation, we assumed that the macromonomers have same reactivity regardless of the ω-functionalization.

The NPs were then transferred into DMF, the solvent used in the grafting step (Experimental part). The sizes of the NPs in DCM/EtOH and in DMF were measured by DLS (Table 2). NPs with diameters of about 350 nm in DCM/EtOH and 250 nm in DMF were obtained. With these results, we proved that with the Vancomycin functionalization, the NPs stay stable. The functionalization has several influences on the NPs size. A decrease of the NPs size after their transfer in DMF was observed. This phenomenon can be explained by a lower polarity of DMF compare to the mixture DCM/EtOH 35:65. By this way, the PEO shell tends to be contracted. This size drop is more marked with NP2, functionalized with Vancomycin, probably because of a


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Biomacromolecules

lower affinity of the Vancomycin with DMF in comparison with the hydroxyl groups of the nude NPs (Figure 1).

Table 2: Characteristics of the functionalized NPs z-average diameters of NPs (nm) macromonomersb

(DLS) and polydispersity index In EtOH/CH2Cl2

In DMF

NP1

(1) and (2)a

310 (0.18)

270 (0.15)

NP2

(2) and (3)a

350 (0.28)

200 (0.13)

a

[(2)]/[(1)]=[(2)]/[(3)]=1/3

b

[Macromonomers]/[Nb] = 0.025


Biomacromolecules

12 z-average diameter: 350 nm (0.277) in EtOH/CH2Cl2 200 nm (0.133) in DMF

10

EtOH/CH2Cl2 N=1 EtOH/CH2Cl2 N=2 EtOH/CH2Cl2 N=3

8 Intensity

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DMF N=1 6

DMF N=2 DMF N=3

4 2 0 10

100

1000

10000

diameter (nm) Figure 1: Distribution profiles of the NPs sizes functionalized with carboxylic acid groups and Vancomycin in the reaction solvent (EtOH/CH2Cl2 mixture) and in DMF. For each solvent, the measure has been carried out three times (N=1, 2, 3) This method allowed us to obtain Vancomycin-functionalized polymeric NPs with high yields of conversion. Another advantage of this macromonomer route is the possibility to synthesize NPs with several functionalities as carboxylic acid function and Vancomycin in a controlled ratio. Knowing the NPs size, it was possible to determine the number of Vancomycin molecules per particle (Nvanco/NP) by using the following equation:

,

!/-.

=

/& ×01 ×21 ×-3 %&

= 1.2 10 9:;;

Vancomycin Functionalized Nanoparticles for Bactericidal Biomaterial

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