Antimicrobial activity of nitric oxide-releasing Ti-6Al-4V metal oxide

Article abstract of DOI:10.3390/jfb8020020

Titanium and titanium alloy materials are commonly used in joint replacements, due to the high strength of the materials. Pathogenic microorganisms can easily adhere to the surface of the metal implant, leading to an increased potential for implant failure. The surface of a titanium-aluminum-vanadium (Ti-6Al-4V) metal oxide implant material was functionalized to deliver an small antibacterial molecule, nitric oxide. S-nitroso-penicillamine, a S-nitrosothiol nitric oxide donor, was covalently immobilized on the metal oxide surface using self-assembled monolayers. Infrared spectroscopy was used to confirm the attachment of the S-nitrosothiol donor to the Ti-Al-4V surface. Attachment of S-nitroso-penicillamine resulted in a nitric oxide (NO) release of 89.6 ± 4.8 nmol/cm² under physiological conditions. This low concentration of nitric oxide reduced Escherichia coli and Staphylococcus epidermidis growth by 41.5 ± 1.2% and 25.3 ± 0.6%, respectively. Combining the S-nitrosothiol releasing Ti-6Al-4V with tetracycline, a commonly-prescribed antibiotic, increased the effectiveness of the antibiotic by 35.4 ± 1.3%, which allows for lower doses of antibiotics to be used. A synergistic effect of ampicillin with S-nitroso-penicillamine-modified Ti-6Al-4V against S. epidermidis was not observed. The functionalized Ti-6Al-4V surface was not cytotoxic to mouse fibroblasts.

Full text of DOI:10.3390/jfb8020020

Journal of
Functional
Biomaterials
Article
Antimicrobial Activity of Nitric Oxide-Releasing
Ti-6Al-4V Metal Oxide
Nina A. Reger , Wilson S. Meng 2 and Ellen S. Gawalt 1,3,
1
*
1
Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA 15282, USA;
regern@duq.edu
Division of Pharmaceutical Sciences, Duquesne University, Pittsburgh, PA 15282, USA; meng@duq.edu
McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15282, USA
Correspondence: gawalte@duq.edu; Tel.: +1-412-396-4709
2
3
*
Academic Editor: Anil Mahapatro
Received: 25 May 2017; Accepted: 16 June 2017; Published: 21 June 2017
Abstract: Titanium and titanium alloy materials are commonly used in joint replacements, due
to the high strength of the materials. Pathogenic microorganisms can easily adhere to the
surface of the metal implant, leading to an increased potential for implant failure. The surface
of a titanium-aluminum-vanadium (Ti-6Al-4V) metal oxide implant material was functionalized
to deliver an small antibacterial molecule, nitric oxide. S-nitroso-penicillamine, a S-nitrosothiol
nitric oxide donor, was covalently immobilized on the metal oxide surface using self-assembled
monolayers. Infrared spectroscopy was used to confirm the attachment of the S-nitrosothiol donor to
the Ti-Al-4V surface. Attachment of S-nitroso-penicillamine resulted in a nitric oxide (NO) release of
2
8
9.6 ± 4.8 nmol/cm under physiological conditions. This low concentration of nitric oxide reduced
Escherichia coli and Staphylococcus epidermidis growth by 41.5
Combining the S-nitrosothiol releasing Ti-6Al-4V with tetracycline, a commonly-prescribed antibiotic,
increased the effectiveness of the antibiotic by 35.4 1.3%, which allows for lower doses of antibiotics
to be used. A synergistic effect of ampicillin with S-nitroso-penicillamine-modified Ti-6Al-4V
against S. epidermidis was not observed. The functionalized Ti-6Al-4V surface was not cytotoxic
to mouse fibroblasts.
±
1.2% and 25.3
±
0.6%, respectively.
±
Keywords: Ti-6Al-4V; nitric oxide; self-assembled monolayers; S-nitroso-penicillamine; Escherichia
coli; Staphylococcus epidermidis
1
. Introduction
Titanium and its alloys have been utilized as a joint replacement material for dental, cardiovascular,
and structural applications, due to strength of the materials, biocompatibility, and corrosion resistance.
Titanium-aluminum-vanadium (Ti-6Al-4V) alloy was developed as an alternative to pure titanium
as it has an increased elastic modulus, yield strength, and ultimate strength, factors important for
the development of a metallic implant material [
hip and knee joints, where hard tissue has failed [
osseointegration is important to reduce implant wear, rejection, and failure [
rejection and failure can also result from infection [ ]. If bacterial adhesion occurs before the tissue
regeneration process after implantation, the host defense cannot prevent the surface colonization
of bacteria, potentially leading to biofilm formation and infection [ ]. Systemic antibiotics are often
delivered in the hours leading up to a surgical procedure, but 90% of all implants show pathogenic
microorganism adhesion post-implantation [ ]. Thus, inhibiting initial bacterial adhesion through
anti-infective implant materials is essential to preventing implant failure and rejection [3].
1
3
,
2
]. Ti-6Al-4V implant materials are utilized in
]. In these applications, tissue regeneration and
]. However, implant
2,4
3,5,6
3
5,7
J. Funct. Biomater. 2017, 8, 20; doi:10.3390/jfb8020020
www.mdpi.com/journal/jfb

J. Funct. Biomater. 2017, 8, 20
2 of 16
The continuous and systemic antibiotic therapies have increased the prevalence of antibiotic
resistance, as over 2 million individuals in the United States annually contract an antibiotic-resistant
infection [
site-specific delivery applications, through the direct modification of nanoparticles and various
biomaterials [ 16]. The small, non-polar nature of NO allows it to easily cross the bacterial cell
8]. A promising antimicrobial alternative, nitric oxide (NO), has been utilized in many
9–
membrane, even in cases of antibiotic-resistant cells where the cell membrane has thickened (i.e.,
methicillin-resistant Staphylococcus aureus (MRSA)) [17]. The antibacterial effect of NO is due to
oxidative and nitrosative stress it causes on the DNA, cell membrane, and proteins of a bacterial
cell [18,19]. Low concentrations of NO have also been shown to trigger biofilm dispersal events and
increase the effectiveness of a low concentration of antibiotic [18,20].
Unfortunately, the short half-life of NO makes delivery and storage difficult [10,21]. Thus, NO is
typically delivered through a donor, such as N-diazeniumdiolates (NONOates) and S-nitrosothiols.
Most nanoparticles and biomaterials are functionalized with NONOates, as the release of NO
from these donors is tailorable and predictable upon protonation. Unfortunately, NONOates have
the potential to decompose into potentially carcinogenic nitrosamines following NO release [22].
S-Nitrosothiols are natural NO donors, as the immune system produces these donors in response to a
bacterial infection. These donors are light- and temperature-sensitive and are, therefore, more difficult
to work with, but offer no harmful side effects or chance of antimicrobial resistance.
NONOate donors have previously been presented at the surface of metallic implants using
physisorption and synthetic polymeric coating methods [11,12,23]. Direct modification of a metallic
surface to release NO was reported by Gallo and Mani [23]. Cobalt-chromium stents were modified
with phosphonoacetic acid, followed by diethylenetriame (DETA)/NONOate. Although these stents
were not tested against bacterial cultures, the modified metal surface released micromolar (
µM)
amounts of NO in vitro 23]. Nablo and coworkers generated a stainless steel implant coated with
[
a silica sol-gel containing a NONOate donor that released picomole amounts of NO over 24 h,
while reducing Staphylococcal bacterial adhesion onto the coated surface [12]. Holt et al. coated
the surface of commercially-pure titanium fixation pins utilizing a silica xerogel containing a NONOate
functionality. Like Nablo and coworkers, these titanium pins released similar amounts of NO and
reduced bacterial adhesion 48 days post implantation in a mouse model [11,12]. Although these
polymeric coatings seem promising, the long-term cytotoxicity of these NONOate silica gel coatings
were not discussed, and could impose limitations on in vivo applications [11,12].
Antimicrobial compounds, such as antibiotics [24–27] and antimicrobial peptides [28,29],
have been presented at the surface of metallic and ceramic biomaterials using self-assembled
monolayers (SAMs) to reduce bacterial adhesion and colonization. SAMs are ordered molecular
assemblies that form spontaneously via chemisorption onto an oxide-rich material and are composed
of a head group, alkyl chain, and a tail group [30
implant surface, the alkyl chain provides stability, and the tail group can be functionalized with
bioactive molecules at the interface [30 31]. Thus, SAMs can be utilized to immobilize NO donors to
,31]. The head group strongly binds to the metal
,
the surface of metallic implant materials. This approach presents bioactive molecules on the surface
while preserving the physical properties of the implanted metal material [32]. These SAMs strongly
adhere to the metal oxide surface and remain through solvent and water rinsing and sonication and
physical tests, including scratch and tape tests [33].
Here, a Ti-6Al-4V metal oxide implant material was modified at the surface to release nanomolar
amounts of NO from a strongly-bound SAM. A reaction at the monolayer terminus provided
an interface for further surface reactions in which an S-nitrosothiol NO donor was immobilized onto
the surface (Scheme 1). Previous immobilization of NO donors to metallic surfaces has been completed
through the use of silica coatings and physisorption. This study immobilizes the S-nitrosothiol donor
at the interface of the metal surface and eliminates the use of silica coatings. Additionally, the use of
S-nitrosothiols avoids the potentially harmful side effects of NONOate donors. The functionalized
Ti-6Al-4V substrates were tested for antimicrobial properties and cytotoxic effects and have the

J. Funct. Biomater. 2017, 8, 20
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potential to deliver an antimicrobial alternative directly to an infection site, limiting the chance of
antibiotic resistance.
2
. Materials and Methods
2
.1. Materials
Titanium-aluminum-vanadium (Ti-6Al-4V composed of 90% titanium, 6% aluminum, and 4%
vanadium) foil of a 0.52 mm thickness was purchased from Goodfellow, Inc. (Corapolis,
PA, USA). 16-Phosphonohexadecanoic acid (COOH-Pa, 97%), N-hydroxysuccinimide (NHS),
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), D-penicillamine (98–101%),
sodium nitrite (NaNO , 99.999% trace metal), Dulbecco’s phosphate-buffered saline (pH = 7.4), and
2
ampicillin sodium salt were purchased from Sigma-Aldrich (Saint Louis, MO, USA) and used as
received. Diethylenetriamine pentaacetic acid (DTPA, >99.0%) was purchased from Fluka (Mexico City,
Mexico). Hydrochloric acid (HCl), 2-propanol (Optima), and tetracycline hydrochloride were obtained
from Fisher Scientific (Waltham, MA, USA), and used as received. Tetrahydrofuran (THF, Optima) was
obtained from Fisher Scientific and was distilled over sodium and benzophenone before use. Ethanol
(
200 proof) and deionized water (ddH O) was obtained from Duquesne University. The nitrate/nitrite
2
colorimetric assay kit was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Lennox
LB Media was obtained from MP Biomedicals, Inc. (Solon, OH, USA) and two capsules were dissolved
◦
®
per 50 mL of ddH O and autoclaved at 121 C before use. Escherichia coli (ATCC 25922), Staphylococcus
2
®
epidermidis (ATCC 14990), and 3T3 Swiss Albino mouse embryo fibroblasts (CCL-92) were obtained
from American Type Culture Collection (ATCC) (Manassas, VA, USA). Dulbecco’s Modified Eagle’s
Medium (DMEM), fetal bovine serum (FBS), penicillin/streptavidin, and Trypsin/EDTA were
purchased from LONZA (Protsmouth, NH, USA). The Live/Dead® Cytotoxicity/Viability Assay
Kit for mammalian cells was purchased from Life Technologies/Fisher Scientific (Waltham, MA, USA).
2
.2. Methods
2
.2.1. Substrate Preparation and Monolayer Formation
Ti-6Al-4V foils were sanded using four decreasing grain size sand papers (150, 320, 400,
and 600 grit) and then cut into 1 cm
×
1 cm square coupons. The coupons were cleaned by sonication
in acetone for 15-min, followed by immersion in boiling methanol for 15 min. The coupons were
◦
placed in a 60 C oven overnight to dry before further use.
SAMs of COOH-Pa were formed on the surface of cleaned Ti-6Al-4V using an aerosol spray
deposition method. A 1 mM solution of COOH-Pa was prepared in distilled THF and was poured
into a thin layer chromatography (TLC) sprayer (Sigma-Aldrich, Saint Louis, MO, USA). The TLC
sprayer was used in conjunction with a stream of nitrogen gas to spray the 1 mM COOH-Pa solution
onto the Ti-6Al-4V coupons in four different cycles. Each coupon was sprayed once with COOH-Pa,
◦
placed in the 60 C oven for 30 min, and then sprayed again with COOH-Pa for a total of four
◦
cycles. The coupons were placed in the oven at 60 C overnight to remove excess solvent before film
characterization. The coupons were rinsed and sonicated with THF to test the mechanical stability of
the formed SAM.
2
.2.2. Immobilization of Nitric Oxide Donor
S-nitroso-penicillamine, the NO donor, was immobilized onto the Ti-6Al-4V metal oxide surface
using a two-step approach (Scheme 1a). Coupons with stable COOH-Pa monolayers were immersed in
a solution containing 20 mM EDC and 50 mM NHS in 2-propanol [24 34]. The reaction proceeded with
,
nitrogen bubbling through the solution for 1.5 h, protected from light. The coupons were removed
from solution and dried under vacuum (0.1 Torr) for 45 min before further reaction.

J. Funct. Biomater. 2017, 8, 20
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S-nitroso-penicillamine was generated by reacting D-penicillamine with acidified nitrite. Briefly,
00 mM of penicillamine was reacted with 100 mM NaNO and 250 mM HCl in the presence of 100
1
µM
2
DTPA (a metal chelator) in an ice bath for 1 h and 15 min, while protected from light (Scheme 1b) [35,36].
After RSNO formation, the S-nitroso-penicillamine solution was diluted with 200 proof ethanol.
The activated COOH-Pa coupons were immersed in the ethanol solution for 1.5 h, with nitrogen
bubbling through. The coupons were removed from the solution without a solvent meniscus and
placed under vacuum (0.1 Torr) overnight to dry.
O
O
OH
NH
N
O
O
O
O
ONS
O
ONS
OH
O
OH
EDC
NH2
(
a)
9
OH
N
9
9
O
O
P
P
P
O O O OH
O O O OH
O O O OH
Ti-6Al-4V Oxide
Ti-6Al-4V Oxide
Ti-6Al-4V Oxide
O
O
(
b)
HS
ONS
NaNO , HCl, DTPA
2
OH
OH
0oC
NH
2
NH
2
Scheme 1.
(a) The two-step reaction sequence for the immobilization of S-nitroso-penicillamine
to the surface of COOH-Pa modified Ti-6Al-4V. The carboxylic acid group is first activated
with ethylenediaminecarbodiimide (EDC)/N-hydroxysuccinimide (NHS) and then reacted with
S-nitroso-penicillamine to form an amide bond. (b) The formation of S-nitroso-penicillamine from the
reaction of acidified nitrite with penicillamine in the presence of a metal chelator.
.2.3. Characterization Techniques
2
Ultraviolet-Visible Spectrometric Analysis
Liquid S-nitroso-penicillamine was scanned from 800 nm to 250 nm using a Varian
ultraviolet-visible (UV-VIS) spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) to
determine the wavelength of maximum absorbance and the purity of the formed S-nitrosothiol
compound. S-nitroso-penicillamine was diluted 1:1 with ddH O to reduce signal saturation.
2
Infrared Spectroscopy
SAM formation and stability, as well as the activation and immobilization steps of S-nitroso-
penicillamine on Ti-6Al-4V, was characterized using a Nexus 470 Fourier Transform Infrared (FTIR)
spectrometer (Nicolet Instruments, Madison, Wisconsin, USA) with a diffuse reflectance infrared
Fourier transform (DRIFT) attachment. Each sample was analyzed under inert conditions with
− −1
1
2
56 scans (4000–400 cm ) and a 4 cm resolution. Clean, unmodified Ti-6Al-4V was used as a
background before sample collection.
In Vitro Nitric Oxide Release
NO release from S-nitroso-penicillamine-modified Ti-6Al-4V was monitored over 48 h using
the Griess assay [23
added to three separate vials containing 900
,
37]. Briefly, three coupons of S-nitroso-penicillamine-modified Ti-6Al-4V were
◦
µL of PBS and placed on an incubator/shaker at 37 C,
while protected from light. The addition of PBS begins the decomposition process of S-nitrosothiols,

J. Funct. Biomater. 2017, 8, 20
5 of 16
as it contains low concentrations of metals that trigger NO release [38]. At each time point, the PBS was
◦
collected and frozen at
−
20 C until assay preparation. The same volume of PBS that was removed at
each time point was replaced [23].
The nitrate/nitrite colorimetric assay included reagents that allowed for the construction of a
nitrate/nitrite calibration curve. Briefly, 80
L of enzyme cofactor mixture and 10
L of Griess Reagent R1 and 50 L of Griess Reagent R2 were added to each well. The well plate was
incubated at room temperature for 10 min before being read using an Infinite M1000 microplate reader
Tecan, Morrisville, NC, USA) at an absorbance of 540 nm. The concentration of NO was determined
by the calibration curve and equation provided by Cayman Chemical (n = 9) [39].
µL of the thawed NO/PBS sample was incubated with
10
µ
µL of nitrate reductase mixture. After incubation for 1 h,
5
0
µ
µ
(
2
.2.4. Bacterial Turbidity
Bacterial optical density was used to monitor the effectiveness of NO release from Ti-6Al-4V
coupons against planktonic cultures of E. coli and S. epidermidis (ATCC, Manassas, VA, USA) in
Lysogeny broth (LB) media. An overnight culture of E. coli or S. epidermidis was diluted to an OD600nm
of 0.1. In each well of a 24-well plate, 100 µL PBS, and 900 µL of planktonic OD600nm E. coli or
S. epidermidis culture was added. Three wells each contained: (i) E. coli/S. epidermidis, (ii) E. coli/
S. epidermidis + unmodified Ti-6Al-4V, or (iii) E. coli/S. epidermidis + S-nitroso-penicillamine-modified
Ti-6Al-4V. The plate was placed in a shaker incubator (230 rpm for S. epidermidis reduces bacterial
◦
adhesion to the surface of Ti-6Al-4V) at 37 C for 2.5 h while protected from light. After incubation,
the OD600nm was collected for each well using a Varian UV-VIS spectrophotometer with three replicates
per well (n = 9). LB media containing PBS was utilized as the blank.
2
.2.5. Antibiotic Susceptibility
The effectiveness of S-nitroso-penicillamine-modified Ti-6Al-4V in conjunction with commonly-
prescribed antibiotics, tetracycline and ampicillin, were tested [18
used was 100 ng/mL and used in experiments involving E. coli. Ampicillin at a concentration of
00 ng/mL was utilized in experiments involving S. epidermidis. Both antibiotic concentrations were
previously determined in the laboratory and were chosen because of the ability to slightly inhibit
bacterial growth, not completely inhibit it [18 20]. An overnight culture of E. coli or S. epidermidis was
diluted to a 0.1 OD600nm. To each of three wells, 900 L of diluted planktonic E. coli or S. epidermidis
culture was placed in each well of a 24-well plate, with: (i) E. coli/S. epidermidis + 100 L PBS,
L tetracycline/ampicillin + unmodified Ti-6Al-4V or (iii) E. coli/
,40]. The concentration of tetracycline
4
,
µ
µ
(ii) E. coli/S. epidermidis + 100 µ
S. epidermidis + 100
µ
L tetracycline/ampicillin + S-nitroso-penicillamine-modified Ti-6Al-4V. The plate
◦
was incubated for 2.5 h at 37 C under shaking conditions (230 rpm for S. epidermidis cultures). The
bacterial turbidity of each well was collected using the Varian UV-Vis spectrophotometer at 600 nm,
with three replicates of each well and LB media with the antibiotic/PBS subtracted as the blank (n = 9).
2
.2.6. In Vitro Cytotoxicity of NO-Releasing Ti-6Al-4V
The cytotoxicity of S-nitroso-penicillamine-modified Ti-6Al-4V was determined against NIH3T3
Swiss Albino mouse embryo fibroblasts. The cells were grown in DMEM supplemented with 10%
FBS and 1% penicillin/streptavidin. The fibroblasts were cultured until approximately 80% confluent.
The cells were trypsinized from the flask, counted, and diluted to a concentration of 10,000 cells per mL
of fresh DMEM. Three of each type of coupon, either unmodified or S-nitroso-penicillamine-modified
Ti-6Al-4V, was placed in a well of a 24 well plate and 1 mL of cell suspension was added. The plate
◦
was incubated for 24 h at 37 C in a 5% carbon dioxide environment.
After a 24 h incubation, the number of live and dead fibroblast cells on the Ti-6Al-4V surfaces was
®
determined using a Live/Dead Viability/Cytotoxicity Kit. Five spots on each microscope slide with
2
an area of 0.6 mm were imaged under 10
×
magnification using an Axioskop2 fluorescent microscope.
The number of live and dead cells was counted on 45 images for each sample and control (n = 45).

J. Funct. Biomater. 2017, 8, 20
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The percent viability for each Ti-6Al-4V sample was calculated by dividing the number of live cells by
the total number of cells (live and dead) and was normalized to the fibroblast control [41].
2
.2.7. Statistics
Statistical analysis was performed using Origin 8.0 software. Where applicable, the Analysis of
Variance (ANOVA) with a Bonferroni post-hoc test was used to determine the averages, standard
errors, and statistical significance of the collected data at the p < 0.05 level of significance. All analyzed
data is presented as the mean ± standard error and all experiments were repeated at least twice.
3
3
3
. Results
.1. Characterization of S-Nitroso-Penicillamine-Modified Ti-6Al-4V
.1.1. Self-Assembled Monolayer Formation
Methylene stretching peaks for an ordered monolayer are
ν
≤
2918 cm 1 and
−
CH2 asymmetric
−
1
ν
≤
2850 cm
[31,33,42,43]. The methylene stretching region of the DRIFT spectrum
CH2 symmetic
of COOH-Pa on Ti-6Al-4V contains two peaks corresponding to the asymmetric and symmetric
−
1
CH stretches, at 2917 and 2848 cm , respectively (Figure 1a). These peaks are indicative of a
2
well-ordered film on the metal surface, characterized by alkyl chains in an all trans, crystalline-like
conformation [20,31,33]. Additionally, the methylene stretches remain after solvent rinse and sonication,
indicating a stable monolayer of COOH-Pa on the surface of Ti-6Al-4V [31].
The binding mode of COOH-Pa to the native oxide surface of Ti-6Al-4V was also examined
using DRIFT (Figure 1b). When using organic acids containing termini of both carboxylic and
phosphonic acid functional groups, the SAM preferentially forms with phosphonic acid as the head
group and carboxylic acid group free at the tail [24,31,44]. The binding mode is determined by
comparing solid COOH-Pa to that of the COOH-Pa monolayer on Ti-6Al-4V [24]. The spectrum of solid
−
1
COOH-Pa contains peaks at 1214, 1077, 1008, 951, and 934 cm corresponding to vP=O, vP-O asymmetric
,
vP-O symmetric, vP-OH, and vP-OH of the phosphonic acid head group, respectively. The DRIFT spectrum
−
1
of COOH-Pa bound to the Ti-6Al-4V surface contains one broad peak at 1085 cm for vP-O. The loss of
both P-OH peaks and the P=O peak, in combination with one broad P-O stretch, suggests a tridentate
binding mode. However, a slight shoulder on the broad vP-O peak could suggests some hydrogen
bonding between the oxygen atom of the P=O stretch and the hydrogen of a surface hydroxyls,
indicating an overall mixture of bidentate and tridentate binding is likely occurring (Figure 1c) [24].
1
00.0
1
00
1
00
9
9
9
9
9
9
9.8
9.6
9.4
9.2
9.0
8.8
9
9
9
9
8
7
9
9
8
8
5
0
5
0
1
076
931
949
2
848
1211
1006
1
085
96
95
94
1
mM COOH-Pa Deposition
75
COOH-Pa on Ti-6Al-4V, Sonication
COOH-Pa Solid
2917
THF Rinse
THF Sonication
7
0
3
000
2950
2900
2850
2800
2750
2700
1300
1200
1100
1000
900
-
1
-1
Wavenumbers (cm
)
Wavenumbers (cm )
(
a)
(b)
Figure 1. Cont.

J. Funct. Biomater. 2017, 8, 20
7 of 16
O
O
OH
OH
9
9
P
P
O
H O
O O O OH
O
O
OH
Ti-6Al-4V Oxide
(
c)
Figure 1.
region of COOH-Pa on the surface of Titanium-Aluminum-Vanadium (Ti-6Al-4V) indicates an ordered,
stable self-assembled monolayer (SAM) through sonication in solvent. ( ) DRIFT spectra of the
(a) Diffuse Reflectance Infrared Spectroscopy (DRIFT) spectra of the methylene stretching
b
−
1
phosphonic acid head binding region, where the broad peak at 1085 cm with a slight shoulder in the
spectrum of the binding region of COOH-Pa confirms a mixture of bidentate and tridentate binding.
(
c) Illustration of the binding of the SAM to the oxide surface.
3
.1.2. Immobilization of S-Nitroso-Penicillamine
UV-VIS spectroscopy was used to confirm the successful synthesis of S-nitroso-penicillamine,
prior to surface immobilization. Absorbance maxima at 340 nm and 595 nm are indicative of the
electronic transitions of π → π* and nN → π*, respectively, for the SNO functionality in pure, unbound
S-nitroso-penicillamine (Figure 2) [45–47]. Confirmation of this pure synthesis allows for further
reaction, while the lack of the nitrite and nitrous acid signatures from the NO donor indicates that no
excess nitrite or nitrous acid was present in the samples. The lack of interfering species is critical for
the success of the NO release assay and the system itself.
0
0
0
0
0
0
0
0
0
.8
.7
.6
.5
.4
.3
.2
.1
.0
S
-Nitroso-Penicillamine Liquid
340
5
92
3
00
350
400
450
500
550
600
650
Wavelength (nm)
Figure 2. UV-VIS spectrum of the unbound S-nitroso-penicillamine liquid NO donor. The wavelength
of the maximum absorbance at 342 nm and secondary absorbance at 595 nm confirms successful
S-nitrosothiol formation.
The immobilization of S-nitroso-penicillamine to the carboxylic acid tail of COOH-Pa was
completed using carbodiimide coupling. The formation of a succinimidyl ester at the carboxylic
acid tail indicates successful leaving group formation. The following peaks confirm the attachment
−
1
−1
for the NHS ester, 1781 cm for
C=O
of NHS to the carboxylic acid tail of COOH-Pa: 1813 cm for
ν
−
1
ν
for the symmetric stretch of the succinimidyl ester within the ring, and 1740 cm
asymmetric stretch of the succinimidyl ester within the ring (Figure 3a) [48]. The stretch at 1210 cm
ν
for the
C=O
C=O
−
1

J. Funct. Biomater. 2017, 8, 20
8 of 16
is that of
ν
of the ester group [48]. Incomplete activation of the COOH-Pa carboxylic acid tails at the
C-O
−
1
−1
48]. Additionally, the stretch at 1556 cm
surface can be observed by the peak at 1722 cm for
ν
[
C=O
is that of an unreactive side product, N-acylurea [48]. The formation of this product is difficult to
prevent under ambient conditions [48]. After this activation step, the phosphonic acid head group
−
1
remains bound the surface in a primarily tridentate manner, based on the P-O stretch at 1075 cm
The attachment of S-nitroso-penicillamine to the surface was completed through the formation of
an amide bond. S-nitroso-penicillamine immobilization was confirmed by the presence of the
.
ν
C=O
−
1
−1
amide II stretch at 1575 cm (Figure 3b). The peak at
N-H
amide I stretch at 1648 cm and the
ν
−
1
1
730 cm is that of carboxylic acid present in the S-nitroso-penicillamine NO donor, while the stretch
−
1
at 1510 cm for
ν
−
indicates that there is NO on the surface of Ti-6Al-4V. The methylene stretching
peaks at 2913 cm and 2847 cm indicate that the COOH-Pa SAM is still adhered to the Ti-6Al-4V
metal oxide surface.
N=O
1
−1
1
648
100
1
00.0
1813
1556
1210
99
1575
1781
9
9
9
9.5
9.0
8.5
1722
1
730
9
9
9
8
7
6
1510
1
073
1
740
2847
2
913
EDC/NHS Activation of COOH-Pa on Ti-6Al-4V
2
000
1800
1600
1400
1200
1000
800
3
000
2500
2000
1500
1000
-
1
Wavenumbers (cm )
-1
Wavenumbers (cm )
(
a)
(b)
Figure 3.
−1
surface of the COOH-Pa monolayer on Ti-6Al-4V. Stretches for ester formation at 1813 cm , 1781 cm ,
(a) DRIFT spectrum of EDC and NHS activation of carboxylic acid groups presented at the
−
1
−
1
and 1740 cm indicated successful activation of the tail group for immobilization by amide bond
formation. ( ) DRIFT spectrum of the attachment of S-nitroso-penicillamine to the COOH-Pa modified
Ti-6Al-4V surface as confirmed by the presence of amide I (1648 cm ) and amide II (1575 cm ).
b
−
1
−1
−
1
−1
The NO group is present at 1510 cm and the stretch at 1730 cm indicates free carboxylic acid from
the S-nitroso-penicillamine donor.
3
.2. Quantitation of NO Release from S-Nitroso-Penicillamine-Modified Ti-6Al-4V
The total amount of NO released from the surface of the modified Ti-6Al-4V was determined
using the Griess assay, a nitrate/nitrite colorimetric assay. NO degrades rapidly into nitrate under
physiological conditions; this assay converts nitrates to nitrites, and the total amount of nitrite
was quantified to reflect the amount of NO released [23]. The total amount of NO released by
2
S-nitroso-penicillamine-modified Ti-6Al-4V cumulatively over 48 h was 89.6
±
4.8 nmol/cm (Figure 4).
The NO was burst-released between the first and second hour of the assay, followed by a slow release
of NO for the remainder of the assay.

J. Funct. Biomater. 2017, 8, 20
9 of 16
100
8
6
4
2
0
0
0
0
0
0
10
20
30
40
50
Time (Hours)
Figure 4. Quantitation of NO release from S-nitroso-penicillamine-modified Ti-6Al-4V, determined
using a nitrate/nitrite colorimetric assay. The modified Ti-6Al-4V coupons released 89.6
nitric oxide (NO)/cm over a 48-h time period. Each data point expresses the average NO release
standard error (SE).
±
4.8 nmol
2
±
3
.3. Antimicrobial Effect of S-Nitroso-Penicillamine-Modified Ti-6Al-4V
3
.3.1. Bacterial Turbidity: E. coli and S. epidermidis
The antibacterial activity of S-nitroso-penicillamine-modified Ti-6Al-4V against Gram-negative
bacteria was assessed by an E. coli challenge using bacterial turbidity. The planktonic growth of
E. coli incubated with Ti-6Al-4V coupons was monitored via optical density at 600 nm (OD600nm).
After incubating a 0.1 OD600nm E. coli culture with Ti-6Al-4V coupons for 2.5 h, the OD600nm was
collected for all E. coli controls and samples. The normalized average OD600nm was 1.00
±
0.002
for the E. coli control, 0.989 0.007 for the unmodified Ti-6Al-4V control, and 0.585 0.011 for
±
±
S-nitroso-penicillamine-modified Ti-6Al-4V (Figure 5a). The OD600nm of the E. coli incubated with
S-nitroso-penicillamine-modified Ti-6Al-4V was statistically lower than both the E. coli control and
E. coli grown with unmodified Ti-6Al-4V coupons.
S-nitroso-penicillamine modified substrates resulted in a 41.5
the E. coli control. This confirms that S-nitroso-penicillamine is an active antimicrobial compound after
being immobilized to the Ti-6Al-4V metal oxide surface. Additionally, E. coli grown with and without
Ti-Al-4V coupons were statistically the same, indicating that E. coli cells are not adhering to the surface
of the Ti-6Al-4V coupons.
E. coli growth in the presence of
1.2% growth reduction compared to
±
The antibacterial activity of S-nitroso-penicillamine-modified Ti-6Al-4V surfaces against
S. epidermidis, the Gram-positive bacteria, was assessed by a bacterial turbidity challenge. The OD600nm
was collected for all S. epidermidis controls and NO modified Ti-6Al-4V samples after a 2.5 h incubation.
The average OD600nm was 1.00
Ti-6Al-4V control, and 0.747
The OD600nm of the S. epidermidis incubated with S-nitroso-penicillamine-modified Ti-6Al-4V substrates
was statistically lower than both the S. epidermidis control and S. epidermidis grown with unmodified
±
0.005 for the S. epidermidis control, 0.985
0.005 for S-nitroso-penicillamine-modified Ti-6Al-4V (Figure 5b).
±
0.006 for the unmodified
±
Ti-6Al-4V, resulting in a 25.3
The OD600nm of S. epidermidis and S. epidermidis grown with unmodified Ti-6Al-4V were statistically
±
0.6% growth reduction compared to the S. epidermidis control.
the same, indicating that no bacterial cells were adhering to the Ti-6Al-4V surface.

J. Funct. Biomater. 2017, 8, 20
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Figure 5.
Ti-6Al-4V shown as the mean
with unmodified Ti-6Al-4V substrates were not statistically different (#). E. coli inhibition by S-nitroso-
penicillamine-modified Ti-6Al-4V is statistically different (*) than all other groups. ( ) Bacterial turbidity
(a
) Bacterial turbidity assay of E. coli cultures treated with S-nitroso-penicillamine-modified
±
standard error, p < 0.05. Escherichia coli (E. Coli) and E. coli grown
b
assay of Stapyloccocus epidermidis (S. epidermidis) cultures treated with S-nitroso-penicillamine-modified
Ti-6Al-4V. S. epidermidis and S. epidermidis grown with unmodified Ti-6Al-4V substrates were not
statistically different (#), while S. epidermidis grown with S-nitroso-penicillamine-modified Ti-6Al-4V
was statistically different (*) than all other groups.
3
.3.2. Antibiotic Susceptibility
The synergistic effect of the nanomolar amounts of NO released from the Ti-6Al-4V surface
was tested in conjunction with 100 ng/mL tetracycline against E. coli. After 2.5 h, the normalized
average OD600nm for E. coli grown without tetracycline was 1.00 0.002, E. coli grown with 100 ng/mL
tetracycline and unmodified Ti-6Al-4V had an OD600nm of 0.541 0.001, and the average OD600nm
for E. coli cultures in the presence of S-nitroso-penicillamine-modified Ti-6Al-4V and tetracycline was
±
±
0
.349 ± 0.007 (Figure 6a). The OD600nm of E. coli incubated with S-nitroso-penicillamine-modified
Ti-6Al-4V and tetracycline was statistically lower than the E. coli control and E. coli treated
with tetracycline. The addition of S-nitroso-penicillamine Ti-6Al-4V increases the effectiveness of
tetracycline by 35.4 ± 1.3% and, thus, a synergistic effect was observed.
The NO released from S-nitroso-penicillamine-modified Ti-6Al-4V increased the effectiveness
of an antibiotic, tetracycline, against E. coli. The same effect was tested against S. epidermidis
in the presence of ampicillin, an antibiotic effective against Gram-positive bacteria. After 2.5 h
,
.
−
4
the normalized average OD600nm for S. epidermidis without ampicillin was 1.00
S. epidermidis grown with 0.4 g/mL ampicillin and unmodified Ti-6Al-4V had an OD
.542 ± 0.01 and the average OD600nm for S. epidermidis grown with S-nitroso-penicillamine-modified
Ti-6Al-4V and ampicillin was 0.559 0.003 (Figure 6b). The OD600nm of S. epidermidis incubated
±
5.0
×
10
of
00nm
µ
6
0
±
with S-nitroso-penicillamine-modified Ti-6Al-4V and ampicillin was not statistically different than
S. epidermidis cultures grown with unmodified Ti-6Al-4V and ampicillin, indicating no synergistic
effect against a Gram-positive bacterial species.

J. Funct. Biomater. 2017, 8, 20
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Figure 6.
tetracycline and S-nitroso-penicillamine-modified Ti-6Al-4V, shown as mean
E. coli treated with S-nitroso-penicillamine-modified Ti-6Al-4V and tetracycline is statistically different
*) than all other groups. ( ) Antibiotic susceptibility challenge of S. epidermidis cultures treated
with 0.4 g/mL ampicillin and S-nitroso-penicillamine-modified Ti-6Al-4V. S. epidermidis grown with
(a) Antibiotic susceptibility challenge in the presence of E. coli cultures treated with 100 ng/mL
±
standard error, p < 0.05
.
(
b
µ
S-nitroso-penicillamine-modified Ti-6Al-4V and ampicillin and S. epidermidis grown with unmodified
Ti-6Al-4V and ampicillin were not statistically different (#).
3
.4. Cytotoxic Effect of S-Nitroso-Penicillamine-Modified Ti-6Al-4V
The cell viability of NIH3T3 fibroblasts grown on S-nitroso-penicillamine-modified Ti-6Al-4V was
®
determined using a Live/Dead Viability/Cytotoxicity assay. Live and dead cells were counted on each
microscope image and the average percent viability was calculated [41]. The resulting percent viabilities
were normalized to the fibroblasts grown on unmodified Ti-6Al-4V. The normalized average percent
cell viability for fibroblasts grown on unmodified Ti-6Al-4V and S-nitroso-penicillamine-modified
Ti-6Al-4V was 100.0
±
1.3% (
±
Standard Error (SE)) and 101.9
±
1.6%, respectively (Figure 7).
Fibroblasts grown on S-nitroso-penicillamine-modified Ti-6Al-4V had statistically the same percent
cell viability as the unmodified Ti-6Al-4V control, indicating that functionalization of the surface to
release NO did not cause the Ti-6Al-4V substrates to be cytotoxic to mammalian cells.

J. Funct. Biomater. 2017, 8, 20
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Figure 7. Normalized average percent cell viability of NIH3T3 mouse embryo fibroblasts grown on
unmodified Ti-6Al-4V and S-nitroso-penicillamine-modified Ti-6Al-4V. Data is shown as the mean
±
SE, p < 0.05. Fibroblast growth was not statistically different between Ti-6Al-4V and NO-releasing
Ti-6Al-4V (#). The Live/Dead images of NIH3T3 fibroblasts are below, from top to bottom: fibroblasts
grown on unmodified Ti-6Al-4V and fibroblasts grown on S-nitroso-penicillamine-modified Ti-6Al-4V.
4
. Discussion
NO-releasing compounds have not been previously covalently immobilized onto the surface
of metal oxide implant materials, but have been incorporated into polymeric matrices on the metal
surface [11 12 23]. Most publications involving NO release from biomaterials, whether metallic, silicate,
or polymeric, utilize potentially carcinogenic NONOate chemistry [ 12 21 23 49 51]. Here, the release
,
,
9–
,
,
,
–
of NO from a covalently-immobilized S-nitrosothiol donor from the surface of Ti-6Al-4V to reduce
E. coli and S. epidermidis growth and increase the effectiveness of an antibiotic was investigated.
This approach has the potential to be utilized in implant surgeries to reduce the colonization of bacteria
post-implantation and ultimately, prevent implant rejection.
The surface of Ti-6Al-4V was first modified with a COOH-Pa SAM. The phosphonic acid functional
group was bound to the surface of Ti-6Al-4V using the thin oxide layer present at the metal surface.
The methylene stretches of the alkyl chain in the DRIFT spectrum indicate that the SAM is ordered,
giving optimal, crystalline packing of molecules on the surface (Figure 1a). This monolayer was also
stable since it survived rinsing and sonication in solvent, which tests the mechanical and chemical
stability. COOH-Pa was bound to the surface in a mixture of bidentate and tridentate binding, as seen
−
1
by a broad stretch at 1085 cm in the DRIFT spectrum with a shoulder indicating possible hydrogen
bonding with the P=O of the head group with the oxide surface (Figure 1b) [24].
Since COOH-Pa was bound to the surface using the phosphonic head group, the carboxylic acid
tail groups were available for carbodiimide coupling with the primary amine functionality within
S-nitroso-penicillamine. S-nitroso-penicillamine contains both a primary amine and carboxylic acid
functionality; therefore, completing the coupling reaction in a single mixture would have likely caused
amide bond formation between individual S-nitroso-penicillamine molecules, and not to the surface
carboxylic acid groups presented from COOH-Pa. As a result, the COOH-Pa modified Ti-6Al-4V
required EDC/NHS activation before reacting with the NO donor. The tail of the COOH-Pa SAM was
activated with EDC and NHS to present NHS at the surface as a more effective leaving group during
−
1
amide bond formation. Activation was confirmed by the presence of stretches at 1813 cm for
ν
C=O

J. Funct. Biomater. 2017, 8, 20
13 of 16
−
1
for the NHS ester, 1781 cm for
ν
for the symmetric stretch of the succinimidyl ester within the
C=O
−
1
ring, and 1740 cm
ν
for the asymmetric stretch of the succinimidyl ester within the ring in the
C=O
DRIFT spectrum (Figure 3a).
The synthesis of the S-nitrosothiol donor was confirmed using UV-VIS, where the wavelength
of maximum absorbance at 340 nm matches that of the characteristic SNO group of other well
characterized S-nitrosothiol donors (Figure 2) [46,47,52]. The UV-VIs spectrum of unbound
S-nitroso-penicillamine lacks contributions from excess nitrite and nitrous acid from the synthesis step
and, thus, could be utilized in further reactions and NO release assays [53].
Reaction of the EDC/NHS activated COOH-Pa modified Ti-6Al-4V coupons with S-nitroso-
−
1
−1
penicillamine showed two stretches in the infrared spectrum at 1648 cm and 1575 cm indicative
of amide I and amide II bond formation, respectively, indicating successful S-nitroso-penicillamine
−
1
attachment to the surface (Figure 3b). Additionally, the NO moiety at 1510 cm in the infrared
−
1
spectrum further confirm attachment to the metal oxide surface. The peak at 1730 cm is a multiplet
and is due to the unreacted carboxylic acid group of S-nitroso-penicillamine and unreacted carboxylic
acid groups at the SAM termini. The N-acylurea present after the activation step does not appear in
the infrared spectrum of S-nitroso-penicillamine attachment, indicating that the unreactive product
−
1
was likely physisorbed onto the surface of the metal. The multiple small peaks within the 1730 cm
stretch could also indicate some unreacted carboxylic acid groups from the COOH-Pa presented at the
metal surface, as observed during the activation step.
The amount of NO released from the surface of S-nitroso-penicillamine-modified Ti-6Al-4V was
determined through the Griess assay, which measures the amount of NO as it is converted from nitrate
2
to nitrite. From this assay, it was determined that 89.6
±
4.8 nmol of NO was released per cm (Figure 4).
This amount of NO is less than the
µ
M levels of NO reported by Gallo et al. [23], but more than the
2
pmole/cm s amounts reported by Nablo et al. [12] and Holt et al. [11]. S-nitroso-penicillamine is a
tertiary NO donor, which is the most stable of the S-nitrosothiol donors, and is likely responsible for
the slow, increasing release of NO over the 48-time period.
Unbound S-nitroso-penicillamine was found to exhibit inhibitory effects against both E. coli
and S. epidermidis. When immobilized, S-nitroso-penicillamine maintained its antimicrobial activity
like the original, unbound molecule. The nanomolar amount of NO released from S-nitroso-
penicillamine-modified Ti-6Al-4V had a bacteriostatic effect against both E. coli and S. epidermidis
cultures, as observed from the results of the bacterial turbidity assay. E. coli growth was inhibited
by 41.5
significantly different than the E. coli controls (Figure 5a). S-nitroso-penicillamine-modified Ti-6Al-4V
inhibited S. epidermidis growth by 25.3 0.6%, which is significantly different than the S. epidermidis
±
1.2% when grown in the presence of S-nitroso-penicillamine-modified Ti-6Al-4V, which is
±
controls. S-nitroso-penicillamine is not as bacteriostatic against S. epidermidis when compared to E. coli,
but Gram-positive bacteria, in general, are more difficult to inhibit with NO (Figure 5b) [54].
S-nitroso-penicillamine-modified Ti-6Al-4V showed a synergistic effect against E. coli when
coupled with 100 ng/mL tetracycline, resulting in a 35.4
±
1.3% reduction in growth when compared
to E. coli grown with tetracycline (Figure 6a). NO is thought to change the physiology of Gram-negative
cells, making them more susceptible to antibiotic treatment [18]. The NO-releasing film on the metal
oxide developed here is capable of increasing the effectiveness of an antibiotic and, thus, less antibiotic
would be needed to treat an E. coli infection, subsequently reducing the risk of antibiotic resistance.
Unfortunately, S-nitroso-penicillamine-modified Ti-6Al-4V did not exhibit a synergistic effect
against S. epidermidis when coupled with ampicillin or other antibiotics (data not shown) (Figure 6b).
Gram-positive bacteria produce their own nanomolar amounts of NO through bacterial nitric oxide
synthase as a defense mechanism to oxidative stress, antimicrobial treatments, and other bacterial
species [54]. NO production in a Gram-positive bacterial culture increases as the cells are treated with
an antibiotic [54]. Thus, the exogenously-delivered nanomolar amounts of NO from the Ti-6Al-4V
surface in the presence of ampicillin does not inhibit bacterial growth because the bacteria within the
culture are already making their own NO [54].

J. Funct. Biomater. 2017, 8, 20
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To consider this S-nitrosthiol-modified Ti-6Al-4V as a metallic implant material, the metal
surface should not be cytotoxic to mammalian cells in vitro. Though S-nitroso-penicillamine-modified
Ti-6Al-4V inhibits bacterial cells, the modified Ti-6Al-4V should not inhibit mouse fibroblast growth.
S-nitroso-penicillamine-modified Ti-6Al-4V showed no cytotoxic effect to fibroblasts after 24 h, as the
percent viability of fibroblasts grown on S-nitroso-penicillamine-modified Ti-6Al-4V and unmodified
Ti-6Al-4V were statistically the same (Figure 7).
5
. Conclusions
A S-nitrosthiol donor, S-nitroso-penicillamine, was immobilized to the surface of a Ti-6Al-4V
metal oxide implant material using SAMs and carbodiimide coupling. The thin oxide layer on the
surface of Ti-6Al-4V was reacted with an organic acid to form a functional monolayer on the metal
oxide surface. The organic acid presented a carboxylic acid functional group at the surface, allowing
for amide bond formation between the primary amine of S-nitroso-penicillamine. The S-nitrosothiol
modified Ti-6Al-4V released nanomolar amounts of NO in vitro and was bacteriostatic against E. coli
and S. epidermidis, indicating that the antimicrobial activity of S-nitroso-penicillamine was maintained
after immobilization to the Ti-6Al-4V surface. S-nitroso-penicillamine-modified Ti-6Al-4V increased
the effectiveness of tetracycline against E. coli, potentially allowing for the use of lower concentrations
of antibiotics. The same synergistic effect in conjunction with ampicillin against S. epidermidis was not
observed, as Staphylococci species are capable of producing their own NO when treated with antibiotics.
Acknowledgments: This work was supported by the Pittsburgh Foundation Hunkele Dreaded Disease Fund.
Author Contributions: N.A.R., W.S.M., and E.S.G. conceived and designed the experiments; N.A.R. performed
the experiments; N.A.R., W.S.M., and E.S.G. analyzed the data; W.S.M. contributed to the bacterial assay materials;
and N.A.R., W.S.M., and E.S.G. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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