PAPER www.rsc.org/polymers | Polymer Chemistry

Dow

nloa

ded

on 0

5 O

ctob

er 2

012

Publ

ishe

d on

28

Janu

ary

2010

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

9PY

0030

9FView Online / Journal Homepage / Table of Contents for this issue

Antimicrobial polysiloxane polymers and coatings containing pendantlevofloxacin

Alex Kugel,a Bret Chisholm,*ab Scott Ebert,b Michael Jepperson,b Laura Jarabekb and Shane Stafslienb

Received 20th October 2009, Accepted 28th December 2009

First published as an Advance Article on the web 28th January 2010

DOI: 10.1039/b9py00309f

A broad-spectrum antimicrobial drug, levofloxacin, was successfully incorporated into a siloxane

coating by covalent attachment. First, an epoxy-functional poly(dimethylsiloxane) (Ep-PDMS) was

synthesized by platinum-catalyzed hydrosilylation using poly(methylhydro-co-dimethyl)siloxane and

allyl glycidyl ether. Next, levofloxacin was reacted with Ep-PDMS using a stoichiometric excess of

epoxy groups relative to levofloxacin to produce a siloxane copolymer containing both pendant

levofloxacin and epoxy moieties (levo-Ep-PDMS). Since attachment (i.e. tethering) of levofloxacin

occurred via ring-opening of epoxy groups by the carboxylic acid group of levofloxacin, the tether

produced was an ester-functional tether. Crosslinked surface coatings were produced by solution

blending the polymer with diethylenetriamine as a crosslinker. Compared to a control coating

produced by simply blending levofloxacin into a polysiloxane, the coating containing tethered

levofloxacin moieties displayed a uniform distribution of levofloxacin, higher initial kill, and

sustained antimicrobial surface activity.

Introduction

In today’s society, advanced medical treatment involves an

increasing number of procedures in which foreign materials are

placed inside or in contact with the human body. For example,

from 1996 to 2001, the number of hip and knee replacement

surgeries increased by 14%.1 Other devices, such as venous and

urethral catheters, are used daily. Whether temporary or

permanent, implantation of these foreign objects into the body

can facilitate transmission of microbial pathogens and cause

infection in patients receiving medical treatment. The annual

infection rate of implant-associated infections in the United

States alone is approaching 1 million per year.2

Multiple strategies have been investigated in an attempt to

inhibit implant-associated infections. The approaches can be

divided coarsely into systemic and local methods. Local methods

of treatment provide the advantages of higher drug concentra-

tions at or near the site of implantation3 and improved drug

selection that allows use of some drugs that are ineffective or

suffer toxicity when used systemically.4

Local antimicrobial prophylaxis can be generally divided into

several subgroups: skin antisepsis, antimicrobial irrigation,

antimicrobial carriers, dipping implants in antimicrobial solu-

tions prior to placement in the body, and the antimicrobial

coating of implants.3 The difference between these methods is the

amount of time that the area remains antimicrobial. For long

term solutions, antimicrobial carriers and antimicrobial coatings

aDepartment of Coatings and Polymeric Materials, North Dakota StateUniversity, 1735 NDSU Research Park Drive, Fargo, ND, 58103, USA.E-mail: Bret.chisholm@ndsu.edu; Fax: +1 701 231 5325; Tel: +1 701231 5328bCenter for Nanoscale Science and Engineering, North Dakota StateUniversity, 1805 NDSU Research Park Drive, Fargo, ND, 58105, USA

442 | Polym. Chem., 2010, 1, 442–452

remain effective on the order of weeks to months while the other

methods remain effective less than one day.3

Release of active materials from polymeric systems has been

extensively studied.5–10 Several interrelated mechanisms drive the

release process including polymer erosion, polymer swelling, and

diffusion. These mechanisms are dependent on a variety of

material properties such as porosity, glass transition temperature

(Tg), crystallinity, molecular weight, hydrophilic/hydrophobic

balance, hydrolytic stability, and crosslink density. The release

rates of antimicrobial coatings and antimicrobial-doped poly-

mers typically tend to follow first-order kinetics with a very

strong release rate initially followed by an exponential decrease

with time.11,12 Several experimental studies involving different

antimicrobials and a broad range of polymers have been

explored yielding well-known commercial products such as the

MR-catheter, a catheter coated on the inner and outer surfaces

with minocycline and rifampine marketed as Cook Spectrum�catheter (Cook Critical Care, USA), and poly-

methylmethacrylate–gentamicin bone cement and implantable

polymeric beads marketed under the Septopal� name.11–13 A

variety of other material/antimicrobial combinations have been

developed to effectively inhibit implant-associated infection.14

These current technologies all include the diffusion-controlled

release of an active ingredient from the system resulting in

a relatively short service lifetime. To extend service lifetime,

covalent attachment (i.e. ‘‘tethering’’) of the active ingredient to

a component of the coating system using a hydrolytically labile

tether is often employed. Since some preferred drugs and poly-

mer carriers may be incompatible, tethering can also offer the

added advantage of forcing compatibility and uniform disper-

sion of the drug throughout the polymer matrix.

One class of antimicrobial compounds that has traditionally

been used systemically to combat infections is the quinolones.

These materials are a class of compounds originating from

This journal is ª The Royal Society of Chemistry 2010

Fig. 1 Several antimicrobial fluoroquinolones generated from the parent compound, nalidixic acid.

Dow

nloa

ded

on 0

5 O

ctob

er 2

012

Publ

ishe

d on

28

Janu

ary

2010

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

9PY

0030

9F

View Online

a by-product of anti-malarial research in 1962 known as nalidixic

acid.15 Through an incremental process over the last 40 years,

nalidixic acid derivatives have grown to be an important class of

antimicrobial chemicals including the well-known antimicrobial

drugs, norfloxacin, ciprofloxacin, ofloxacin, levofloxacin, and

moxifloxacin (Fig. 1).15–19 Ciprofloxacin, which is effective

toward Gram-negative bacteria as well as some resistant strains

of bacteria, was introduced in the mid-1980s. Further develop-

ments led to levofloxacin, an advanced, broad-spectrum

antibiotic with strong activity toward a variety of pathogenic

Gram-negative and Gram-positive bacteria. Levofloxacin is

typically prescribed for a wide range of infections and is the only

respiratory fluoroquinolone approved by the US FDA for the

treatment of nosocomial pneumonia.

While fluoroquinolones are an effective class of antibiotics,

side effects and limitations are known. Some of the most effective

derivatives have been restricted or withdrawn from use due to

their toxicity, and there is evidence that bacterial resistance can

lead to treatment failure.15,16 There is also great concern that

continued widespread use of the most common fluo-

roquinolones, ciprofloxacin and levofloxacin, will result in the

creation of bacterial strains that are resistant to the entire class of

fluoroquinolones.

While a variety of polymers have been used for biomedical

applications, polysiloxanes and other silicon-based materials,

due to their low cytoxicity and desirable physical properties, have

been used quite extensively.20–27 Silicones, however, have low

surface energy and are hydrophobic leading to poor compati-

bility with hydrophilic drugs. Hydrophilic modifiers

This journal is ª The Royal Society of Chemistry 2010

(i.e. channeling agents), such as polyethylene glycol28 or inor-

ganic salts,29 are often necessary for adequate release. The

incorporation of these additives decreases the mechanical prop-

erties and overall lifetime of the coating or device.

As a means to inhibit the formation of resistant strains of

bacteria and eliminate negative side effects associated with

systemic treatment, the authors investigated the feasibility of

producing polysiloxane-based materials that provide local

treatment with levofloxacin. Potential applications for poly-

siloxane materials that provide local treatment with levofloxacin

include coatings for surgical implants,30–33 especially orthopedic

devices, which are prone to infection by the Staphylococcus

family.

Experimental

Materials

Poly(methylhydro-co-dimethyl)siloxane (HMS-501) (900–

1200 g mol�1) and platinum–divinyltetramethyldisiloxane

complex in xylene (Karstedt’s catalyst) were obtained from

Gelest, Inc. (Morrisville, PA). Allyl glycidyl ether, toluene,

glacial acetic acid, 33% hydrogen bromide in acetic acid, crystal

violet, chloroform, potassium hydrogen phthalate (KHP), levo-

floxacin, tetrahydrofuran (THF), methanol, and diethylenetri-

amine (DETA) were purchased from Sigma-Aldrich

(Milwaukee, WI). Intergard 264 was purchased from Interna-

tional Paint LLC (Houston, TX) and DC3140, a silicone coating,

was purchased from Dow Corning (Midland, MI). Mueller

Polym. Chem., 2010, 1, 442–452 | 443

Dow

nloa

ded

on 0

5 O

ctob

er 2

012

Publ

ishe

d on

28

Janu

ary

2010

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

9PY

0030

9F

View Online

Hinton (MH) agar was purchased from VWR International

(West Chester, PA). The respiratory dye, 2,3,5-triphenylte-

trazolium chloride (TTC), was obtained from Amresco (Solon,

OH). Phosphate buffered saline (PBS) was obtained from EMD

chemicals (Gibbstown, NJ) as a 10� PBS liquid concentrate and

diluted to 1� for use in coating preconditioning work by adding

appropriate amounts of purified water. All other materials were

used as received.

Polymer synthesis

An epoxy-functional poly(dimethylsiloxane) copolymer

(Ep-PDMS) was synthesized as follows: a magnetic stirring bar,

50 g toluene, 20.0 g (0.148 equivalents Si–H) poly(methylhydro-

co-dimethyl)siloxane, 16.9 g (0.148 equivalents allyl) allyl gly-

cidyl ether, and 30 mL platinum–divinyltetramethyldisiloxane

complex in xylene were added to a 250 mL round-bottom flask

equipped with a condenser and nitrogen purge. The reaction

mixture was heated to 80 �C and reaction progress monitored

using proton nuclear magnetic spectroscopy (1H NMR) by

observing disappearance of the Si–H peak present at 4.8 ppm.

Solvent was removed at reduced pressure on a rotary evaporator

and the viscous liquid product was collected. The polymer

product was characterized by 1H NMR and an epoxy equivalent

weight end-group titration (ASTM D1652). The polymer

produced possessed an epoxy equivalent weight of 272 g eq�1.

A polysiloxane copolymer containing both pendant epoxy

groups and levofloxacin moieties (levo-Ep-PDMS) was synthe-

sized as follows: 1.8 g (0.0066 equivalents epoxy) Ep-PDMS,

0.09 g (2.5 � 10�4 mol) levofloxacin, 2.0 g methanol, and

a magnetic stir bar were added to an 8 mL scintillation vial and

the stirred reaction mixture heated with a 70 �C silicone oil bath

for 22 h. The product was characterized by NMR, Fourier

transform infrared spectroscopy (FT-IR), high performance

liquid chromatography (HPLC), gel-permeation chromatog-

raphy (GPC), and differential scanning calorimetry (DSC).

Coating preparation

A polysiloxane coating solution based on levo-Ep-PDMS was

produced by blending 1.9 g (0.0064 equivalents epoxy) of levo-

Ep-PDMS in 2.0 g methanol and 138 mL (0.0064 equivalents

N–H) DETA in an 8 mL scintillation vial using a Vortex-Genie

lab mixer by Scientific Industries (Bohemia, NY). A polysiloxane

coating solution containing levofloxacin as an additive was

produced by blending 0.025 g (6.9 � 10�5 mol) levofloxacin,

0.51 g (0.0019 equivalents epoxy) Ep-PDMS, 750 mL chloroform,

and 42 mL (0.0019 equivalents N–H) DETA in an 8 mL scintil-

lation vial using the Vortex-Genie lab mixer. A PDMS coating

solution free of levofloxacin was produced by solution blending

0.5 g (0.0019 equivalents epoxy) Ep-PDMS, 750 mL chloroform,

and 42 mL (0.0019 equivalents N–H) DETA in an 8 mL scintil-

lation vial using the Vortex-Genie lab mixer.

Specimens for characterization were prepared by depositing

coating solutions onto a substrate, allowing the solvent to flash at

ambient conditions for 1 h, and curing the coatings at 50 �C for

22 h. The method of deposition and substrate varied with the

characterization method used. Details are specified in the forth-

coming ‘‘Material characterization’’ section.

444 | Polym. Chem., 2010, 1, 442–452

Material characterization

NMR spectra were collected using a JEOL ECA400 400 MHz

spectrometer equipped with a 24-place autosampler carousel.

Polymer samples were measured in deuterated chloroform using

16 scans for proton spectra and a pulse width of 14.6 ms,

acquisition time of 2.18 s, pulse angle of 45�, attenuation of 6 dB,

pulse time of 7.3 ms, receiver gain of 22, relaxation delay of 4 s,

and repetition time of 6.18 s. Carbon spectra were obtained in

deuterated chloroform with 14 000 scans and a pulse width of

10.9 ms, acquisition time of 1.04 s, pulse angle of 30�, attenuation

of 9 dB, pulse time of 3.6 ms, receiver gain of 50, relaxation delay

of 2 s, and repetition time of 3.04 s. In addition, carbon spectra

were run with decoupling and nuclear overhauser effect (NOE)

activated at an NOE time of 2 s. Distortionless enhancement by

polarization transfer (DEPT) spectra were obtained with

decoupling using 17 000 scans and a pulse width, acquisition

time, and attenuation equivalent to those used for carbon

spectra. Irradiation attenuation was 6 dB with a pulse width of

14.6 ms. Receiver gain was set at 60 and selection angle was 135�.

Heteronuclear multiple quantum coherence (HMQC) was

collected using 112 scans. X-Axis (proton) settings included

acquisition time of 0.14 s, attenuation of 6 dB, and pulse width of

14.6 ms. Y-Axis (carbon) settings included acquisition time of

14.97 ms, attenuation of 9 dB, and pulse width of 10.9 ms.

Decoupling was active, relaxation delay was 1.5 s, and T1 was

1 ms with a repetition time of 1.64 s. JEOL Delta software and

predictions with ChemDraw (CambridgeSoft) software were

used to make peak assignments.

A Bruker Optics Vertex 70 FT-IR was used to collect spectra

in transmission from samples deposited on a silicon 96-spot array

plate. Spectra were analyzed using OPUS software from Bruker.

Polymer molecular weight data were obtained using a Symyx

RapidGPC� which consists of a dual-arm liquid handling robot

coupled to a temperature-adjustable GPC system using an

evaporative light scattering detector (Polymer Laboratories ELS

1000) and 2�PLgel mixed-B columns (10 mm particle size). THF

was used as the eluent at a flow rate of 2.0 mL min�1 and

molecular weights were determined using the aforementioned

column and detector at 45 �C by comparing to polystyrene

standards.

DSC was performed using a Q2000 from TA Instruments

equipped with a liquid nitrogen cooling accessory and 50-place

autosampler. After calibration using an indium standard,

samples were run in a standard heat–cool–heat protocol from

�180 �C to 100 �C at a heating and cooling rate of 10 �C min�1.

Tgs were taken as the inflection point of the second heating cycle

using TA Universal Analysis software.

Water contact angle was measured on coatings produced by

solution casting into a stamped 400 � 800 aluminium panel34 using

a Symyx integrated contact angle measurement device. The

instrument places 10 mL droplets on the coating surface and

captures images using a charge-coupled device (CCD) camera for

subsequent image analysis and contact angle determination. In

addition to static measurements, the instrument was also used to

measure water contact angle hysteresis. For water contact angle

hysteresis, a 10 mL drop of water is placed on the coating surface

and water is added at a constant rate of 0.1 mL s�1 and contact

angle measured at 10 s intervals for 1 min. After 1 min, water is

This journal is ª The Royal Society of Chemistry 2010

Dow

nloa

ded

on 0

5 O

ctob

er 2

012

Publ

ishe

d on

28

Janu

ary

2010

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

9PY

0030

9F

View Online

removed at 0.1 mL s�1 and contact angle measured at 10 s

intervals. Contact angle hysteresis is then calculated by averaging

the first three advancing and the last three receding contact

angles and subtracting the receding average from the advancing

average.

Thermal stability was characterized using a Q500 thermogra-

vimetric analyzer from TA Instruments (New Castle, DE). Using

a standard platinum pan, samples were ramped from ambient to

1000 �C at 20 �C min�1. Nitrogen was used as both the balance

gas (40 mL min�1) and the sample gas (60 mL min�1) with an air-

cool time of 25 min between samples.

Optical images were collected on coatings deposited on glass

cover slips using an Olympus BX61 epifluorescence microscope,

and images were collected with a DP70 12.5 million pixel digital

camera. Fluorescence images were obtained using fluorescence

excitation with a green filter for 20 ms.

The antimicrobial activity of coatings on aluminium discs pre-

coated with an epoxy primer (Intergard 264) and silicone-based

tiecoat (Dow Corning 3140) was determined by placing the

coated discs in 20 mL scintillation vials containing 5 mL of 1�PBS for 24 h at ambient conditions. The 1� PBS solution was

removed and the scintillation vials, containing the coating discs,

were placed in a desiccator for 4 h to facilitate drying of the

coating surfaces. The coating discs were then inverted and placed

on a MH agar plate inoculated with �107 cells mL�1 of the

Gram-negative bacterium, Escherichia coli ATCC 12435. TTC

was incorporated into the MH agar (70 mg L�1) to facilitate

visualization of bacterial growth on the coating surfaces (red

color). Agar plates with inverted coating discs were incubated for

24 h at 37 �C and examined for both zones of growth inhibition

and inhibition of growth on the coating surfaces. The coating

discs were then removed from the MH agar plates and cleaned by

gently wiping the coating surfaces with a moist Kimwipe�. The

cleaned coating discs were placed into new 20 mL scintillation

vials containing 5 mL of fresh 1� PBS for an additional 6 days

(total of 7 days immersion) at ambient laboratory conditions and

evaluated for antimicrobial activity using the agar plating

method described above. This process was repeated several times

to determine the antimicrobial activity of the coating discs to 81

days of total immersion in 1� PBS.

Results and discussion

Polymer synthesis and characterization

As shown in Fig. 2, the synthesis of levo-Ep-PDMS was a two

step process in which glycidyl ether groups were first grafted to

a poly(methylhydro-co-dimethyl)siloxane copolymer using

hydrosilylation chemistry to produce Ep-PDMS. A portion of

the epoxides present on Ep-PDMS was reacted with levofloxacin

to produce levo-Ep-PDMS. NMR was used to characterize levo-

Ep-PDMS as well as the polymer intermediate, Ep-PDMS. Fig. 3

displays NMR spectra obtained for Ep-PDMS along with peak

assignments. Peak assignments were determined using the four

spectral techniques shown in Fig. 3. Using integration of proton

peaks, the number of protons corresponding to each peak was

determined. Carbon spectra provided the carbon peak infor-

mation. DEPT135 was used to distinguish primary, secondary,

tertiary and quaternary substituted carbon atoms. This

This journal is ª The Royal Society of Chemistry 2010

technique provides spectra in which methyl (primary) and

methine (tertiary) peaks orient in one direction, while methylene

(secondary) carbon peaks orient in the opposite direction. Tetra-

substituted (quaternary) carbon peaks do not appear in the

DEPT135 spectrum. By comparing to a carbon spectrum,

assignments are easily made. HMQC is a two-dimensional

technique that correlates proton and carbon peak information.

A peak is observed when protons and carbons are associated

with one another. From HMQC spectra, complex proton spectra

can be assigned through correlation with the appropriate carbon

atoms. In the 1H NMR, the presence of the peaks corresponding

to the protons of the methylene group (‘‘o’’ in Fig. 3) and the lack

of a peak at 4.8 ppm corresponding to silicon hydride protons

demonstrate successful grafting of glycidyl ether groups to the

polysiloxane backbone. The formation of pendant levofloxacin

groups by reaction of carboxylic acid groups of levofloxacin with

glycidyl groups of Ep-PDMS was demonstrated by the obser-

vation of peaks in both the 1H NMR and 13C NMR spectra that

correspond to the isopropyl group generated by ring opening of

the glycidyl ether groups of Ep-PDMS (Fig. 4). Levofloxacin has

been previously characterized by NMR.35

The reaction of levofloxacin with Ep-PDMS was also char-

acterized using FT-IR (Fig. 5). Levofloxacin has been charac-

terized previously by FT-IR.36 Briefly, bands in the levofloxacin

spectrum are as follows: 3268 cm�1 (OH), 2959–2803 cm�1 (CH3),

1722 cm�1 (C]O acid), 1622 cm�1 (C]O ring). Bands for Ep-

PDMS are as follows: 2959–2803 cm�1 (–CH3, –CH2–),

1259 cm�1 (Si–CH3), 1193 cm�1 (Si–CH2–), 1090 cm�1

(–Si(CH3)2–O–), 1026 cm�1 (–Si(CH3)2–O–), 911 cm�1 (epoxy

ring), 846 cm�1 (Si–CH3), and 800 cm�1 (Si–CH3). For the

reaction product, levo-Ep-PDMS, an increase in the OH band at

3411 cm�1 can be seen which was due to the formation of the

secondary hydroxyl group resulting from the epoxy ring-opening

reaction. In addition, evidence for the formation of the ester

tether was obtained by comparing the carbonyl region (1722

cm�1 to 1592 cm�1) of spectra obtained before and after reaction.

The reaction resulted in a large reduction in the band for the

carboxylic acid carbonyl of levofloxacin at 1722 cm�1. Also

observed was a relative increase in the carbonyl band at 1622

cm�1 and the formation of a new carbonyl band at 1592 cm�1.

These changes in the carbonyl bands suggest the formation of the

ester tether via epoxy ring-opening by the levofloxacin carboxylic

acid group. Although significantly reduced in intensity, the

existence of the carboxylic acid carbonyl band at 1722 cm�1

indicated the presence of some unreacted levofloxacin in the

polymer sample.

GPC was also used to characterize the reaction of levofloxacin

with Ep-PDMS. Fig. 6 compares GPC chromatograms for Ep-

PDMS, levofloxacin, a physical blend of Ep-PDMS and levo-

floxacin corresponding to the reaction mixture used to prepare

levo-Ep-PDMS, and the reaction product, levo-Ep-PDMS. The

chromatogram obtained for levo-Ep-PDMS lacks the peak

centered at 525 s which is associated with Ep-PDMS. In addition,

the single peak obtained for levo-Ep-PDMS was shifted to higher

retention time and the molecular weight distribution was nar-

rowed as compared to Ep-PDMS. These results provide further

evidence that levofloxacin was successfully tethered to

Ep-PDMS. The reduction in retention time and narrowing of the

molecular weight distribution obtained by tethering of

Polym. Chem., 2010, 1, 442–452 | 445

Fig. 2 Synthetic scheme used to produce epoxy-functional levofloxacin-containing polysiloxane copolymers.

Dow

nloa

ded

on 0

5 O

ctob

er 2

012

Publ

ishe

d on

28

Janu

ary

2010

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

9PY

0030

9F

View Online

levofloxacin to Ep-PDMS suggest that attachment of the levo-

floxacin molecule to the polysiloxane as a side chain results in

intramolecular interactions in THF that reduce the hydrody-

namic volume of the polymer.

The thermal properties of Ep-PDMS and levo-Ep-PDMS were

determined using DSC and TGA. As shown in Fig. 7, Ep-PDMS

was an amorphous polymer exhibiting a single Tg at�89 �C. The

reaction of Ep-PDMS with levofloxacin to produce levo-Ep-

PDMS resulted in an 18 �C shift in Tg consistent with the bulky

446 | Polym. Chem., 2010, 1, 442–452

levofloxacin moiety significantly reducing segment mobility of

the polysiloxane polymer backbone.

Fig. 8 provides a comparison of the thermal stability of Ep-

PDMS, levofloxacin, and levo-Ep-PDMS. Levo-Ep-PDMS

shows approximately a 5% weight loss between 140 �C and the

onset of the primary decomposition process at 325 �C. This

weight loss is most likely due to the loss of absorbed

water resulting from the hydrophilic nature of the levofloxacin

moieties.

This journal is ª The Royal Society of Chemistry 2010

Fig. 3 NMR characterization of Ep-PDMS.

Dow

nloa

ded

on 0

5 O

ctob

er 2

012

Publ

ishe

d on

28

Janu

ary

2010

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

9PY

0030

9F

View Online

Coating characterization results

A surface coating, which will be referred to as ‘‘tethered,’’ was

prepared from levo-Ep-PDMS by solution blending with DETA

which served as a crosslinker for the pendant epoxy groups. For

comparison purposes, two other coatings were produced which

will be referred to as ‘‘control’’ and ‘‘doped.’’ The control coating

was Ep-PDMS crosslinked with DETA while the doped coating

was basically the control with 5 wt% levofloxacin as an additive

in the coating, i.e. the levofloxacin was not chemically attached to

This journal is ª The Royal Society of Chemistry 2010

the coating matrix. Fig. 9 displays images of the three coatings

deposited on glass cover slips. The opacity observed for the

doped coating clearly indicated that the levofloxacin was insol-

uble in the polysiloxane matrix. Covalently linking, i.e. tethering,

the levofloxacin to the polymer matrix prevented macroscopic

phase separation as indicated by the transparent coating

produced.

Further characterization of the morphology of these coatings

was conducted using both optical and fluorescence microscopy.

Polym. Chem., 2010, 1, 442–452 | 447

Fig. 4 NMR characterization of levo-Ep-PDMS.

Dow

nloa

ded

on 0

5 O

ctob

er 2

012

Publ

ishe

d on

28

Janu

ary

2010

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

9PY

0030

9F

View Online

Since levofloxacin fluoresces strongly, tethered and doped

samples were produced using 1.0 wt% levofloxacin as opposed

to 5.0 wt% to facilitate morphological characterization. As

shown in Fig. 10, the levofloxacin moieties were uniformly

distributed throughout the tethered coating while the fluores-

cence for the doped coating was non-uniform due to insolu-

bility or limited solubility of the levofloxacin in the coating

matrix.

As shown in Fig. 11, the Tg of the control coating and doped

coating was the same (�85 �C) while that of the tethered coating

448 | Polym. Chem., 2010, 1, 442–452

was 40 �C higher due to limitations on polymer chain segmental

mobility induced by the bulky levofloxacin moieties. The equiv-

alence of the Tg obtained for both the control coating and doped

coating indicated that reaction of levofloxacin with the epoxy

groups of Ep-PDMS did not occur to any significant extent

during curing of the coating at 50 �C. While the Tg of the tethered

coating was significantly higher than that of a typical crosslinked

PDMS elastomer, it was still well below room temperature and,

as a result, exhibited the rubbery character desired for many

biomedical applications.

This journal is ª The Royal Society of Chemistry 2010

Fig. 5 FT-IR spectra used to characterize the levofloxacin tethering reaction: levofloxacin; Ep-PDMS; physical mixture of Ep-PDMS with levofloxacin;

and levo-Ep-PDMS.

Fig. 6 GPC chromatograms showing molecular weight distribution

differences between: levofloxacin; Ep-PDMS; physical mixture of

Ep-PDMS with levofloxacin; and levo-Ep-PDMS.

Fig. 7 DSC thermograms for HMS-501, Ep-PDMS, and levo-Ep-

PDMS.

Fig. 8 A comparison of thermal stability by TGA of levo-Ep-PDMS,

Ep-PDMS, and levofloxacin.

Dow

nloa

ded

on 0

5 O

ctob

er 2

012

Publ

ishe

d on

28

Janu

ary

2010

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

9PY

0030

9F

View Online

The high polymer backbone flexibility of PDMS enables the

production of a regular array of methyl groups to be oriented

outward to the air interface resulting in a highly hydrophobic

surface.37 Since surface wettability and water egress into the

coating will strongly affect antimicrobial activity, water contact

angle and water contact angle hysteresis were measured. As

shown in Table 1, both the control coating and the doped coating

exhibited the high water contact angle typically observed for

PDMS, while the tethered coating displayed a significantly lower

water contact angle. The similarity between the contact angle

obtained for the control and doped coatings indicated that the

composition of the doped coating at the very outer surface was

This journal is ª The Royal Society of Chemistry 2010 Polym. Chem., 2010, 1, 442–452 | 449

Fig. 9 Images of the three crosslinked coatings produced. ‘‘Control’’

was produced by crosslinking Ep-PDMS with DETA. ‘‘Tethered’’ was

produced by crosslinking levo-Ep-PDMS with DETA. ‘‘Doped’’

was produced by solution blending levofloxacin, Ep-PDMS, and DETA.

Fig. 10 Images from optical (left) and fluorescence (right) microscopy of

coatings made on glass cover slips containing 1 wt% ‘‘doped’’ (a and c)

and ‘‘tethered’’ (b and d) levofloxacin.

Fig. 11 DSC thermograms of the coatings produced.

Table 1 Water contact angle and water contact angle hysteresis dataobtained for the coatings

Water contact angle/� Contact angle hysteresis

Control 90.48 � 1.11 2.51Doped 94.20 � 2.68 13.01Tethered 84.80 � 1.38 12.47

450 | Polym. Chem., 2010, 1, 442–452

Dow

nloa

ded

on 0

5 O

ctob

er 2

012

Publ

ishe

d on

28

Janu

ary

2010

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

9PY

0030

9F

View Online

essentially polysiloxane methyl groups. Considering the insolu-

bility of the levofloxacin in the coating matrix, it was not

a surprise that the composition of the coating surface would be

largely comprised of the lower surface energy polysiloxane

matrix. Since the levofloxacin moieties present in the tethered

coating are covalently bound to the polysiloxane matrix, phase

separation is limited and polymer backbone mobility hindered.

Due to these factors, the chemical composition of the coating

surface may include some fraction of levofloxacin moieties

resulting in the lower water contact angle.

Contact angle hysteresis (CAH) is a general indicator of

surface chemical or morphological stability.38,39 In general, CAH

can be used as an indication of the degree of surface instability

resulting from wetting of the surface. The control coating had

a low CAH, indicative, in this case, of a surface stable to water-

induced rearrangement. Both the doped and tethered coatings,

on the other hand, showed a large relative CAH, indicative of

surface instability.

Antimicrobial characterization results

Antimicrobial activity of the coatings was determined as a func-

tion of immersion time in PBS using agar plates inoculated with

E. coli. As shown in Fig. 12, the control coating showed extensive

microbial growth on the surface of the coating and no evidence of

a zone of inhibition. In contrast, both the doped and tethered

coatings showed antimicrobial activity on the surface of the

coating and a relatively large zone of growth inhibition. Inter-

estingly, the zone of growth inhibition for the tethered coating was

initially much larger than that for the doped coating indicating

greater levofloxacin release from the tethered coating. This result

may be due to differences in coating morphology and levofloxacin

dispersion as well as the presence of some ‘‘free,’’ i.e. unreacted,

levofloxacin in the tethered coating. The optical and fluorescence

microscopy results showed that the levofloxacin phase separated

from the polysiloxane matrix for the doped coating while no

evidence of macroscopic phase separation was observed for the

tethered coating. Considering the insolubility of levofloxacin in

the polysiloxane matrix and the low surface energy of poly-

siloxanes, the smaller zone of growth inhibition exhibited by the

doped coating may be due to the generation of a coating surface

morphology consisting of a largely polysiloxane-rich composition

at the coating-air interface that is much lower in levofloxacin than

the bulk of the coating. Since the levofloxacin is covalently bound

to the polysiloxane matrix for the tethered coating, levofloxacin

moieties must necessarily be in the vicinity of the coating-air

interface thereby facilitating release from the coating.

After 28 days of immersion in PBS, the doped coating dis-

played no antimicrobial activity while the tethered coating

This journal is ª The Royal Society of Chemistry 2010

Fig. 12 Images of coated specimens preconditioned in PBS for various time periods and tested for antimicrobial activity toward E. coli using an agar

plating method. The ‘‘darkness’’ observed on the surface of the 81 day tethered disc is a result of repeated measurement of the same samples and not due

to growth of E. coli on the surface.

Dow

nloa

ded

on 0

5 O

ctob

er 2

012

Publ

ishe

d on

28

Janu

ary

2010

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

9PY

0030

9F

View Online

showed antimicrobial activity exclusively at the coating surface.

This result suggests that the zone of growth inhibition observed

for the tethered coating before immersion was most likely due to

leaching of residual, unreacted levofloxacin from the coating.

After sufficient leaching of free levofloxacin from the coating,

only tethered levofloxacin moieties remained thus eliminating the

observation of a zone of growth inhibition. The long-term

activity is due to the slow release of levofloxacin through

a hydrolytic mechanism. The presence of some ‘‘free’’ or

unreacted levofloxacin in the tethered coating is consistent with

the FT-IR results discussed earlier in this document.

Conclusion

The broad-spectrum antimicrobial agent, levofloxacin, was

successfully tethered to Ep-PDMS by ring-opening pendant

epoxides to produce an ester-functional tether. Attachment of

levofloxacin to the polysiloxane resulted in a significant increase

in Tg. The levo-Ep-PDMS was then used to produce a cross-

linked coating by reacting pendant epoxides with DETA. Unlike

the doped coating, the tethered coating was a one-phase material

in which the levofloxacin moieties were uniformly dispersed

throughout the coating. With regard to antimicrobial activity,

the tethered coating allowed for a greater initial release of levo-

floxacin and prolonged activity toward E. coli when compared to

the doped coating. The greater initial release of levofloxacin from

the tethered coating was attributed to the more uniform distri-

bution of levofloxacin as well as to the presence of some ‘‘free,’’

i.e. unreacted, levofloxacin. These results suggest that tethering

levofloxacin to a polysiloxane matrix may result in the

This journal is ª The Royal Society of Chemistry 2010

production of new surface coatings to combat implanted device-

related infection.

Acknowledgements

The authors acknowledge the Office of Naval Research under

grant N00014-07-1-1099. Special thanks to Kenneth Johnson

for assistance in statistical design analysis pertaining to these

coatings.

References

1 R. A. Deyo, A. Nachemson and S. K. Mirza, N. Engl. J. Med., 2004,350, 722–726.

2 R. O. Darouiche, N. Engl. J. Med., 2004, 350, 1422–1429.3 O. Darouiche Rabih, Clin. Infect. Dis., 2003, 36, 1284–1289.4 M. Lucke, B. Wildemann, S. Sadoni, C. Surke, R. Schiller,

A. Stemberger, M. Raschke, N. P. Haas and G. Schmidmaier,Bone, 2005, 36, 770–778.

5 S. Conti, L. Polonelli, R. Frazzi, M. Artusi, R. Bettini, D. Cocconiand P. Colombo, Curr. Pharm. Biotechnol., 2000, 1, 313–323.

6 S. Kumar and A. Ali, Indian J. Hosp. Pharm., 1995, 32, 129–133.7 A. Kydonieus, Treatise on Controlled Drug Delivery—Fundamentals,

Optimisation, Applications, Marcel Dekker, New York, 1992.8 Y. Loo and K. W. Leong, Biomaterials for drug and gene delivery, An

Introduction to Biomaterials (Biomedical Engineering), ed. S. A.Guelcher and J. O. Hollinger, CRC Press, Boca Raton, FL, 2005,pp. 342–368.

9 L. Masaro and X. X. Zhu, Prog. Polym. Sci., 1999, 24, 731–775.10 M. V. S. Varma, A. M. Kaushal, A. Garg and S. Garg, Am. J. Drug

Delivery, 2004, 2, 43–57.11 C. von Eiff, B. Jansen, W. Kohnen and K. Becker, Drugs, 2005, 65,

179–214.12 C. Von Eiff, W. Kohnen, K. Becker and B. Jansen, Int. J. Artif.

Organs, 2005, 28, 1146–1156.

Polym. Chem., 2010, 1, 442–452 | 451

Dow

nloa

ded

on 0

5 O

ctob

er 2

012

Publ

ishe

d on

28

Janu

ary

2010

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

9PY

0030

9F

View Online

13 I. Francolini, G. Donelli and P. Stoodley, Rev. Environ. Sci. Bio/Technol., 2003, 2, 307–319.

14 E. M. Hetrick and M. H. Schoenfisch, Chem. Soc. Rev., 2006, 35, 780–789.

15 F. Van Bambeke, J. M. Michot, J. Van Eldere and P. M. Tulkens,Clin. Microbiol. Infect., 2005, 11, 256–280.

16 P. Ball, Curr. Ther. Res., 2003, 64, 646–661.17 K. F. Croom and K. L. Goa, Drugs, 2003, 63, 2769–2802.18 M. Hurst, H. M. Lamb, L. J. Scott and D. P. Figgitt, Drugs, 2002, 62,

2127–2167.19 A. J. Schaeffer, Am. J. Med., 2002, 113, 45S–54S.20 F. Abbasi, H. Mirzadeh and A.-A. Katbab, Polym. Int., 2001, 50,

1279–1287.21 ACS Symposium Series: Science and Technology of Silicones and

Silicone-Modified Materials, ed. S. J. Clarson, J. J. Fitzgerald,M. J. Owen, S. D. Smith and M. E. Van Dyke, Oxford UniversityPress, New York, NY, 2007, vol. 64, p. 400.

22 R. A. Compton, J. Long-Term Eff. Med. Implants, 1997, 7, 29–54.23 Silicon Containing Polymers: The Science and Technology of Their

Synthesis and Applications, ed. R. G. Jones, W. Ando andJ. Chojnowski, Kluwer Academic Publishers, Norwell, MA, 2000,p. 768.

24 C. R. McMillin, Rubber Chem. Technol., 1994, 67, 417–446.25 C. R. McMillin, Rubber Chem. Technol., 2006, 79, 500–519.26 Biomaterials Science, An Introduction to Materials in Medicine, ed.

B. D. Ratner, A. S. Hoffman, F. J. Schoen and J. E. Lemons,Elsevier Academic Press, London, 2nd edn, 2004, p. 851.

27 R. Yoda, J. Biomater. Sci., Polym. Ed., 1998, 9, 561–626.28 J. Schulze Nahrup, Z. M. Gao, J. E. Mark and A. Sakr, Int. J. Pharm.,

2004, 270, 199–208.

452 | Polym. Chem., 2010, 1, 442–452

29 G. Di Colo, Controlled drug release from implantable matrixes basedon hydrophobic polymers, in Proceedings and Program of the 18thInternational Symposium on Controlled Release of BioactiveMaterials, ed. I. W. Kellaway, Controlled Release Society,Deerfield, IL, 1991, pp. 317–318.

30 H. S. El-Zaim and J. P. Heggers, Silicones for pharmaceutical andbiomedical applications, in Polymeric Biomaterials, ed.S. Dumitriu, Marcel Dekker, Inc., New York, NY, 2nd edn,2002, pp. 79–90.

31 B. Gottenbos, H. C. van der Mei, F. Klatter, P. Nieuwenhuis andH. J. Busscher, Biomaterials, 2002, 23, 1417–1423.

32 T. J. Laing, D. Schottenfeld, J. V. LaceyJr, B. W. Gillespie,D. H. Garabrant, B. C. Cooper, S. G. Heeringa, K. H. Alcser andM. D. Mayes, Am. J. Epidemiol., 2001, 154, 610–617.

33 J. J. H. Oosterhof, K. J. D. A. Buijssen, H. J. Busscher,B. F. A. M. van der Laan and H. C. van der Mei, Appl. Environ.Microbiol., 2006, 72, 3673–3677.

34 B. J. Chisholm, S. J. Stafslien, D. A. Christianson, C. Gallagher-Lein,J. W. Daniels, C. Rafferty, L. Vander Wal and D. C. Webster, Appl.Surf. Sci., 2007, 254, 692–698.

35 G. Fardella, P. Barbetti, I. Chiappini and G. Grandolini, Int. J.Pharm., 1995, 121, 123–127.

36 S. Sagdinc and S. Bayari, THEOCHEM, 2004, 668, 93–99.37 M. J. Owen, in Surface Properties and Applications, ed. R. G. Jones,

W. Ando and J. Chojnowski, Kluwer Academic Publishers,Dordrecht, 2000.

38 P. Majumdar, S. Stafslien, J. Daniels and D. C. Webster, J. Coat.Technol. Res., 2007, 4, 131–138.

39 J. H. Wang, P. M. Claesson, J. L. Parker and H. Yasuda, Langmuir,1994, 10, 3887–3897.

This journal is ª The Royal Society of Chemistry 2010