1

For Table of contents

0

5

10

15

20

25

30

1 3 5

Ga2O3 (mol%)

Dia

met

er (m

m)

E.coli P.aeruginosa S.aureus MRSA C.difficle

Novel quaternary gallium-doped phosphate-based glasses are unique for controlled delivery

of Ga3+ which found to inhibit bacterial growth. The lack of new antibiotics in development

makes these glasses potential new therapeutic agents for pathogenic bacteria including MRSA

and C. difficile.

2

Antimicrobial Gallium-doped Phosphate-based Glasses

Doctor Sabeel P. Valappil1&2, Doctor Derren Ready3, Doctor Ensanya A. Abou Neel1 Doctor

David M. Pickup4, Doctor Wojciech Chrzanowski1,Doctor Luke A. O’Dell 5, Professor

Robert J. Newport4, Professor Mark E. Smith5, Professor Michael Wilson2

and Professor Jonathan C. Knowles1*.

1Division of Biomaterials and Tissue Engineering and 2Division of Microbial Diseases,

University College London, Eastman Dental Institute, 256 Gray’s Inn Road, London.

3Microbiology, Eastman Dental Hospital, UCLH NHS Foundation Trust, 256 Gray’s Inn

Road, London. 4School of Physical Sciences, University of Kent, Canterbury, CT2 7NH,

UK. 5Department of Physics, University of Warwick. Coventry, CV4 7AL, U.K.

*Corresponding Author. Mailing address: Division of Biomaterials and Tissue Engineering,

UCL Eastman Dental Institute, 256 Gray's Inn Road, London WC1X 8LD. UK. Phone: +44

(0)207 915 1189, Fax: +44 (0)207 915 1227, Email:j.knowles@eastman.ucl.ac.uk

3

Abstract

Novel quaternary gallium-doped phosphate-based glasses (1, 3, and 5 mol% Ga2O3) were

synthesized using a conventional melt quenching technique. The bactericidal activities of the

glasses were tested against both Gram-negative (Escherichia coli and Pseudomonas

aeruginosa) and Gram-positive (Staphylococcus aureus, methicillin-resistant Staphylococcus

aureus, and Clostridium difficile) bacteria. Results of the solubility and ion release studies

showed that these glass systems are unique for controlled delivery of Ga3+. 71Ga NMR

measurements showed that the gallium is mostly octahedrally coordinated by oxygen atoms,

whilst FTIR spectroscopy provided evidence for the presence of a small proportion of

tetrahedral gallium in the samples with the highest gallium content. FTIR and Raman spectra

also afford an insight into the correlation between the structure and the observed dissolution

behaviour via an understanding of the atomic-scale network bonding characteristics. The

results confirmed that the net bactericidal effect was due to Ga3+, and a concentration as low

as 1 mol % Ga2O3 was sufficient to mount a potent antibacterial effect. The dearth of new

antibiotics in development makes Ga3+ a potentially promising new therapeutic agent for

pathogenic bacteria including MRSA and C. difficile.

Key words: phosphate-based glasses; gallium content; bactericide; MRSA; C. difficile

4

Introduction

Recently, much attention has been focused on the need for new antimicrobial agents due to

the increased prevalence of antibiotic-resistant bacteria[1]. The multi-resistant nosocomial

pathogens such as MRSA and Clostridium difficile are the main source of recent increases in

the incidence of hospital-acquired infections (HAIs). Latest figures from the Health Protection

Agency (UK) showed that there were 15,592 cases of C. difficile infection reported in

England in the first quarter of 2007 (January to March). This represents a 22% increase on the

count for the previous quarter (October to December 2006). Similarly, the latest MRSA

bloodstream infection figures showed that there were 1,444 cases reported in England within

the period of January 2007 to March 2007 alone. Despite these trends, only one new

antibacterial drug, called linezolid, with a completely novel mechanism of action has been

introduced in the past 3 decades, and very few new antibiotics are in the advanced stages of

development [1]. Hence there is significant scope to develop novel drugs that combat these

antibiotic-resistant bacteria.

Iron (Fe) metabolism is a key factor in vulnerability of infecting bacteria as they require Fe

for growth and the functioning of key enzymes; such as those involved in DNA synthesis,

electron transport and oxidative stress defenses [2]. Therefore smooth functioning of Fe

metabolism is critical in the pathogenesis of bacterial infections. Gallium (Ga3+) has an ionic

radius nearly identical to that of Fe3+ and can function as a Trojan horse as many biological

systems are unable to distinguish Ga3+ from Fe3+ [3]. More importantly, sequential oxidation

and reduction are critical for many of the biological functions of Fe3+. However,

supplementation of Ga3+ can disrupt Fe3+-dependent processes because, unlike Fe3+, Ga3+

cannot be reduced under the same conditions [3]. Gallium is already approved by FDA to treat

hypercalcemia of malignancy [4] and has recently emerged as a new generation antibacterial

ion that may be useful in treating and preventing localized infections [5]. However, the use of

5

gallium as an antimicrobial agent could be significantly improved by the development of an

effective means of delivery. Chemically-durable materials, that can slowly release gallium

ions for long periods, would be considered desirable materials for medical applications.

Phosphate-based glasses (PBGs) are such durable materials which can act as a unique system

for the delivery of metal ions in a controlled way [6]. Ions incorporated into the glass structures

are not a separate phase, and thus their rate of release is defined by the overall degradation

rate of the glass. Metal ions, such as copper and silver, have been incorporated into PBGs, and

these glasses have then been used as wound dressings to prevent infections[7] and also to

control urinary tract infections in patients needing long-term indwelling catheters [7, 8] .

However, there is an underlying need to improve the properties of existing biomaterials due to

the incidence of HAIs, which often lead to revision surgery. Currently, prophylaxis in the

form of systemically administered antibiotics serves as the main weapon against infection

following implant surgery [9]. Therefore the need for improved antimicrobial agents and better

delivery devices could be met by gallium-doped PBGs. So far gallium has been shown to be

effective against the organisms causing syphilis, trypanosomiasis [10] , tuberculosis [11] and

malaria [12] in humans. It is also effective in the treatment of pneumonia in foals caused by

Rhodococcus equi [13].

The aim of this study was to develop novel gallium-doped PBGs and to investigate the

efficacy of these glasses against bacterial pathogens associated with HAIs such as

Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, methicillin-resistant

Staphylococcus aureus (MRSA) and Clostridium difficile.

2. Results and Discussion

2.1. Glass degradation

As can be seen in Figure.1a, the weight loss data decreased with an increase in the Ga2O3

content of the glasses. However, there were no perceptible differences in the profiles of the 3

6

and 5 mol% Ga2O3 compositions until 48hours. The Gallium free PBGs was found to

dissolve completely by 72hours of incubation. The dissolution rates, obtained by applying a

line of best fit through the data, were 41.70, 23.60, 7.30 and 3.70 µg.mm2.hr-1 for the 0, 1, 3

and 5 mol% Ga2O3 compositions respectively.

6

7

8

9

0 24 48 72 96 120 144

Time (h)

pH

0 1 3 5

Figure 1. Dissolution, determined by weight loss, (a) and pH analysis (b) of the 0, 1, 3 and 5 mol% Ga2O3-doped PBGs as a function of time.

y = 0.0417x

R2 = 0.9792

y = 0.0236x

R2 = 0.9866

y = 0.0073x

R2 = 0.9851

y = 0.0037x

R2 = 0.995

0

1

2

3

4

5

6

0 24 48 72 96 120 144

Time (h)

Wei

gh

t lo

ss (m

g.m

m2 )

0 1 3 5

1 (a)

1 (b)

7

The observed reduction in dissolution rate associated with the increase in Ga2O3 content could

be accounted to the associated increase in the ionic strength of the leaching solution. Glass

degradation has been reported to consist of the three synergistic processes of ion exchange,

hydration, and finally hydrolysis of the phosphate chains while in solution [14]. As a result of

ion exchange, a gel layer usually formed on the glass surface, i.e. hydration, and when it

leached into the surrounding medium it causes an increase in the ionic strength of the solution

with the resultant reduction in the dissolution rate.

2.2. pH Analysis

The pH analysis revealed that its value increased as the Ga2O3 content decreased (Figure 1b).

Gallium free composition displayed the maximum increase (8.61) in pH from the initial value

of 7. However, the pH value for both 3 and 5 mol% Ga2O3 remained close to neutral for the

duration of the study. As is seen from Figure 1b, the pH for compositions with 3 and 5 mol%

Ga2O3 remained neutral for the duration of the study, whereas for the composition with 0 and

1 mol% Ga2O3, the pH increased to about 8.5. The hydrolysis of PBGs exhibit clear pH

dependence as Watanabe et al.[15] stated that the rate of hydrolysis of small ring cyclic

trimeta- and tetrametaphosphates decreased in acidic solutions, and increased in basic

solutions with an increase in the pH value for all solvents.

2.3. Ion release

As expected, the highest levels of cation release, calcium (Ca2+) (Figure. 2a) and sodium ions

(Na+) (Figure. 2b), were observed for the composition with the highest dissolution rate, 0 and

1 mol% Ga2O3 containing glasses. The Ca2+ ion release data correlated with that of the

solubility data obtained. For the Ca2+ release profiles (Figure 2a), the gallium-free

composition released the greatest amount of ions. Also, compositions with higher sodium

mol% released more Na+ ions into solution, and it was directly proportional to the solubility

values, suggesting that sodium ions were released into solution first.

8

It can be seen from Figures 2a and b that greater amounts of Na+ ions than Ca2+ ions were

released from the compositions investigated. However, both Na+ and Ca2+ ion release profiles

showed clear differences between the compositions investigated, with a decrease in Na+ and

Ca2+ ion release seen with increasing Ga2O3 mol%. The greatest Na+ release was seen for the

gallium-free composition, with statistically not significant difference seen between the 3 and 5

mol% Ga2O3 compositions up to 48h. This may be expected as the gallium ions (Ga3+) were

added in place of Na2O, therefore, there was less sodium present in the other two

compositions.

The amounts of phosphorous ions released in this study appear to be linear in nature

(Figure.2c). The release of phosphorous ion decreased as the Ga2O3 content increased in the

glasses, with 5mol% Ga2O3 releasing the least amount of phosphorous. The use of ICP-MS

has enabled the detection of the total amount of phosphorous ions, and this method is found to

be superior to ion chromatography where availability of standards restricts the total detection

of different phosphate species.

0

5

10

15

20

25

30

35

40

45

50

55

60

0 24 48 72 96 120Time (h)

Ca

ion

rel

ease

(pp

m)

0 1 3 5

2 (a)

9

0

50

100

150

200

250

0 24 48 72 96 120Time (h)

Na

ion

rel

ease

(pp

m)

0 1 3 5

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

0 24 48 72 96 120Time (h)

P io

n r

elea

se (p

pm

)

0 1 3 5

2 (c)

2 (b)

10

As can be seen from Figure 2d, no Ga3+ was detected from the 0 mol% Ga2O3 composition as

expected, however clear differences are seen between the 1, 3 and 5 mol% Ga2O3

compositions. The 1 mol% Ga2O3 composition released the highest levels of Ga3+ ions, with

the 5 mol% Ga2O3 compositions releasing the least. The release profile of anionic species

mirrored those of the degradation rate, Na+ and Ca2+ ion release.

2.2. Bactericidal tests

This study was conducted to determine an effective range of Ga2O3-doped PBGs that exhibit

bactericidal activity using disk diffusion assay. The zones of inhibition (i.e. zones of no

visible bacterial growth surrounding the disks) were found to be larger in size for the 1mol%

compared to 3 or 5 mol % Ga2O3-doped PBGs when tested against S. aureus, E. coli, P.

aeruginosa, MRSA and C. difficile (Figure.3a).

0

5

10

15

20

25

30

35

40

45

50

55

60

0 24 48 72 96 120Time (h)

Ga

ion

rel

ease

(pp

m)

0 1 3 5

Figure 2. Cumulative ion release (a) calcium, (b) sodium, (c) phosphorous, and (d) gallium as a function of time for 0, 1, 3 and 5 mol% Ga2O3-doped PBGs.

2 (d)

11

In all the experiments, the gallium free PBGs were used as negative controls and the zones of

inhibition presented here is the test values minus that of gallium free PBGs. This result was in

good agreement with the Ga3+ ion release from the glass compositions investigated (Figure

2d). P. aeruginosa was found to be the most susceptible organism to Ga3+. Interestingly,

when gallium free PBGs were tested, a zone of inhibition was seen for S. aureus, MRSA and

C. difficile (data not shown). This could have been due to the change in pH during the glass

degradation as observed in water (Figure.1b).

0

5

10

15

20

25

30

1 3 5

Ga2O3 (mol%)

Dia

met

er (

mm

)

E.coli P.aeruginosa S.aureus MRSA C.difficle

Figure 3. (a) Disc diffusion assay conducted on 0, 1, 3 and 5 mol% Ga2O3-doped PBGs against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA) and Clostridium difficile

3 (a)

12

Initial viable count experiments were conducted on Ga2O3–doped PBGs for P.aeruginosa,

using the most potent Ga2O3 concentrations (1 and 3 mol% Ga2O3) (Figure 3b). Gallium-free

PBGs were used as controls, and initial viable counts were conducted prior to addition of the

PBGs to check the viability of the bacteria. Each point represents the log10 of the mean

number of viable count of three samples. Error bars represent standard deviations. Both

1mol% and 3 mol% Ga2O3–doped PBGs containing glasses showed statistically significant

(p=0.0001) reduction in the log10 of the mean number of viable cells compared to Ga2O3 free

control at 4h (Figure 3b). Moreover, the log10 of the mean number of viable cells on the

1mol% and 3 mol% Ga2O3–doped PBGs (p≤0.0006) displayed maximum effect at 12 h

compared to the control. This effect continued with time as 1mol% and 3 mol% Ga2O3–doped

PBGs, continue to show significant difference (p≤0.0004) to the control at 24h. However,

there were no statistically significant differences (p=0.080) observed between 1mol% and

3mol% Ga2O3–doped PBGs at this time point. These results indicated that the overall killing

Figure 3. (b) The effect of 0, 1 and 3 mol% Ga2O3-doped PBGs on the viability of suspensions of Pseudomonas aeruginosa after 4, 12 and 24h incubation. CFU= colony forming units (mean number of viable cells)

7

7.25

7.5

7.75

8

8.25

4 12 24

Time (h)

Lo

g C

FU

/mm

2

0 1 3

3 (b)

13

of the bacteria beyond this time points is largely due to the nutrient suppression than the

gallium concentration.

Recently, an antimicrobial approach using Ga3+ was reported that targets bacterial Fe3+

metabolism by exploiting the chemical similarities between Fe3+ and Ga3+ [5]. Given the

general ability of Ga3+ to substitute for Fe3+, it could interfere with many Fe-requiring

enzymes, including ribonucleotide reductase, which catalyzes the first step in DNA synthesis

[16]; superoxide dismutase and catalase, which protect against oxidative stress [17]; enzymes

involved in oxidative phosphorylation such as cytochromes; and others. It is also possible that

Ga3+ could act on several targets simultaneously [5], and if this is true, fully defining the

mechanism of action of Ga3+ may be difficult. However, this would also suggest that mutation

of a single intracellular target is unlikely to produce high-level Ga3+ resistance in target

organisms.

In vivo studies reported that the suppression of T cell and macrophage activation by Ga3+

could, however, partially counteract gallium's antimicrobial activities as some dose levels

aggravated progression of tuberculosis in gallium-treated guinea pigs [18]. Therefore, dose

versus response in vivo studies using several different infectious agents would elucidate the

relationship between the antimicrobial and immunomodulating activities of gallium. Results

of such studies would significantly help to determine the therapeutic doses of gallium in

treating infections.

2.3. Structural analysis of the gallium-doped phosphate-based glasses

2.3.1. Thermal analysis

Addition of 1 mol% Ga2O3 in PBGs did not produce a change in Tg; however addition of 3

and 5 mol% Ga2O3 produced a significant increase in Tg as given in Table.1.

14

Increasing Tg temperatures with increasing Ga2O3 mol% was expected as Ga2O3 is known as

a refractory material. This finding suggested that addition of 3 and 5 mol% Ga2O3 resulted in

the formation of more cross-linked glass structure. Such suggestion was also correlated well

with the degradation where the highly cross-linked (5 mol% Ga2O3 containing) glasses

showed lower degradation than gallium-free glasses. Tg is a measure of the bulk as opposed

to parameters such as Tc and Tm which can be used as a measure of a particular phase [19].

Andersson [20] proposed a simpler view of Tg against composition; he stated that the higher

the Na2O content, the lower the Tg. This statement was found to be in accordance with the

values obtained from this investigation as Ga2O3 was used to substitute Na2O.

2.3.2. NMR analysis

Figure 4I shows a stacked plot of the 31P MAS NMR spectra obtained with the frequency

scale expanded to show only the MAS centre bands, and the fitted peak data is given in Table

2. The connectivity of the phosphate network is commonly described by Qn notation, where n

refers to the number of bridging oxygen in the PO43- group. Two peaks are clearly visible at

chemical shifts of around −20 and −5 ppm, representing Q2 and Q1 phosphorous sites

respectively, and Figure 4II shows the fitting of the 0 mol% Ga2O3 spectrum, including fitting

of the spinning sidebands (the spinning sideband intensities were included in calculating the

relative abundance of each Qn species). No Q3 or Q0 sites were observed in any of the spectra.

Glass composition (mol%) Glass code P2O5 CaO Na2O Ga2O3

Density Tg (g.cm-3) (0C)

Ca16Na39P45

Ca16Na38P45Ga1

Ca16 Na36P45Ga3

Ca16 Na34P45Ga5

45 16 39 0 2.59 (±0.01) 341.69 (±0.46) 45 16 38 1 2.61 (±0.01) 341.11 (±2.82) 45 16 36 3 2.61 (±0.02) 359.26 (±1.81) 45 16 34 5 2.66 (±0.01) 369.99 (±0.57)

Table 1. Composition of phosphate-based glasses used in this study with corresponding Density and glass transition temperatures, Tg.

15

The 23Na MAS NMR spectra are shown in Figure 4III(a). The peak moved approximately 1

ppm to a more negative shift as the amount of Ga2O3 increased from 0 to 5 mol%. Figure

4III(b) shows a simulation of the 23Na spectrum of the 0 mol% Ga2O3 sample at two different

fields using a Gaussian distribution in quadrupolar coupling constant CQ to represent the

variation in Na environments present in the sample due to disorder. These simulations

yielded a mean value of CQ = (2.65 ± 0.15) MHz, a FWHM distribution in this parameter of

(2.15 ± 0.15) MHz, and a chemical shift value of δISO = (−3 ± 0.5) ppm. The asymmetry

parameter ηQ was kept at 0 for simplicity, although in reality a distribution in CQ would likely

mean a distribution in this, as well as the isotropic chemical shift. These parameters gave a

good fit to all four 23Na spectra, with only a variation in δISO, which moved 1 ppm downfield

as the Ga2O3 content increased from 0 to 5 mol%. These results imply that although there is

likely to be some association between the sodium and gallium cations, the extent of disorder

of the sodium environment is not significantly affected by the gallium content.

As the mol% of Ga2O3 was increased to 5, the relative abundances of the Q1 and Q2

phosphorous sites remained constant within experimental uncertainty. The 31P MAS NMR

line arising from the Q1 site broadened with increasing Ga2O3 content (Table 2), while the Q2

linewidth remained constant within experimental uncertainty. The chemical shifts of both

sites moved to a more negative value as the Ga2O3 content increased [see Figure 4III(b)], with

a larger change for the Q1 site than for the Q2. This is consistent with a previous study of

Ga2O3-containing sodium phosphate glass [21] and is due to increasing covalency in the

Mol% Q2 Shift Q2 Abundance Q2 Linewidth Q1 Shift Q1 Abundance Q1 Linewidth

Ga2O3 (± 0.2 ppm) / % (± 2 %) (± 0.5 ppm) (± 0.2 ppm) / % (± 2 %) (± 0.5 ppm)

0 −20.2 73 9.5 −3.6 27 8.5

1 −20.4 75 9.6 −4.4 25 9.4

3 −20.8 75 9.7 −6.4 25 10.9

5 −21.0 72 9.9 −7.8 28 11.2

Table 2. 31P MAS NMR fit parameters for the Q1 and Q2 sites.

16

P−O−Ga bonding interaction. A similar trend has been observed in CaO-Na2O-P2O5 glasses

as sodium is replaced by calcium [22]. The fact that the linewidth of the 31P NMR Q1 peak

increased while that of the Q2 remained roughly constant suggests that the gallium cations are

associating with the Q1 chain-end groups and increasing the chemical shift range of these

environments by partially replacing the ionic P−O−Na bonding interactions with more

covalent P−O−Ga bonding.

(ppm) -200 -100 0 100 200

4[I]

4[II]

17

4[III]

4[IV]

Figure 4[I]. 31P MAS NMR spectra obtained (a) 5 mol% Ga2O3, (b) 3 mol% Ga2O3, (c) 1 mol% Ga2O3 and (d) 0 mol% Ga2O3. [II] The fit of the 31P MAS NMR spectrum from the 0 mol% Ga2O3 sample, including the spinning sidebands. [III] (a) The 23Na MAS NMR spectra, and (b) simulation of the 0 mol % Ga2O3 sample at two different fields.[IV] The 71Ga MAS NMR spectra of the samples containing Ga2O3, (a) 5 mol % at 18.8 T, (b) 5 mol % at 14.1 T, (c) 3 mol % at 14.1 T and (d) 1 mol % at 14.1 T. Spectrum (a) took four hours to acquire, (b) and (c) took 24 hours each and spectrum (d) took 48 hours.

18

Figure 4IV shows the 71Ga MAS NMR spectra obtained from the 1, 3 and 5 mol% Ga2O3

samples. Figure 4IV(a) is from the 5 mol% Ga2O3 sample at 18.8 T, and figures 4IV(b),

4IV(c) and 4IV(d) are the 5, 3 and 1 mol% Ga2O3 samples respectively, all at 14.1 T. The

spectra all show a lower signal/noise ratio than the 23Na or 31P spectra due to the lower

amount of gallium present in the sample, the lower natural abundance of the 71Ga isotope

(39.9 % compared with 100 % for both 23Na and 31P), and also the wider lineshape due to

second-order quadrupolar broadening. The relatively large linewidth in these spectra meant

that the spinning sidebands lay very close to the centre-band, although they are not very

clearly visible due to their amplitudes being comparable to that of the noise. Each spectrum

shows a peak centred at approximately −50 ppm. The FWHM linewidth of this peak is

approximately 100 ppm, and for the 5 mol% Ga2O3 sample this width did not appreciably

decrease when the field was increased from 14.1 to 18.8 T. A previous 71Ga MAS NMR

study on Ga2O3-Na2O-P2O5 glasses identified a peak at −60 ppm associated with octahedrally

coordinated gallium and one at 120 ppm due to tetrahedral gallium [23]. This suggests that the

71Ga NMR peak observed here arises from octahedrally coordinated gallium. The fact that the

linewidth does not appreciably decrease between 14.1 and 18.8 T indicates that chemical shift

dispersion is the dominant broadening mechanism at these fields rather than second order

quadrupolar broadening. This suggests a distribution of octahedral gallium sites (i.e. the

gallium exists in a disordered environment). The presence of some tetrahedral gallium cannot

be ruled out since a small peak may be present at around 120 ppm in the spectrum from the

sample containing 5 mol% Ga2O3, but certainly the gallium is mostly octahedral.

2.3.3. Vibrational spectroscopy

Figures 5(I) and 5(II) show the FTIR and Raman spectra of Ga2O3-doped compared to Ga2O3-

free PBGs respectively.

19

1600 1400 1200 1000 800 600 4000.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

νs(O-P-O)

νas

(O-P-O)ν

s(PO

3)-

νas

(PO3)-

νs(PO

2)-ν

as(PO

2)-

(d)

(c)

(b)

Wavenumber / cm-1

Abs

orba

nce

(a)

δ(O-P-O)

1600 1400 1200 1000 800 600 400 200

0

250

500

750

1000

1250

δ(O-P-O)νas

(PO3)-

νas

(PO2)- ν

s(O-P-O)

νs(PO

2)-

Cou

nts

Wavenumber / cm-1

(a)

(b)

(c)

(d)

5 [I]

5 [II]

Figure 5. Structural analysis of the Ga2O3-doped PBGs. [I] FTIR spectra from (a) 0 mol% Ga2O3, (b) 1 mol% Ga2O3, (c) 3 mol% Ga2O3 and (d) 5 mol% Ga2O3. [II] Raman spectra from (a) 0 mol% Ga2O3, (b) 1 mol% Ga2O3, (c) 3 mol% Ga2O3 and (d) 5 mol% Ga2O3.

20

The absorption bands in the vibrational spectra have been assigned according to the literature

[24-27]. The band near 1280 cm−1 is assigned to the asymmetric stretching mode of the two non-

bridging oxygen atoms bonded to phosphorus atoms in the Q2 tetrahedral sites, νas(PO2)–; the

shoulder at 1180 cm−1 is assigned to the symmetric stretch of the same structural unit,

νs(PO2)–. The absorption bands near 1100 and 1000 cm−1 are assigned to the asymmetric and

symmetric stretching modes of chain-terminating Q1 groups (νas(PO3)2– and νs(PO3)

2–),

respectively. The band close to 1100 cm−1 has a component at 1150 cm−1 assigned to Q1 end

groups associated with Na+ ions [25]. The absorption band near 900 cm−1 is assigned to the

asymmetric stretching modes of the P–O–P linkages, νas(P–O–P), and the bands at 770 and

720 cm−1 assigned to the symmetric stretching modes of these linkages, νs(P–O–P). The broad

absorption around 540 cm−1 is attributed to O−P−O deformation modes. The weak shoulder to

the high frequency side of this band at ~610 cm−1, observed in the spectrum from the sample

with the highest gallium content, can be assigned to vibrations localized on GaO4 tetrahedra

[26]. The Raman spectra all exhibit five significant absorption bands. The two most intense

absorptions at 1170 and 670 cm−1 are assigned to νs(PO2)– and νs(P–O–P) modes, respectively.

The weaker bands at 1260 and 1020 cm−1 are due to the antisymmetric νas(PO2) – and νas(PO3)

– vibrations, respectively, and finally, the broad low-energy band with contributions at 380

and 340 cm−1 is attributed to O−P−O deformation modes.

The vibrational spectra shown in Figures 5(I) and 5(II) are quite typical of those from PBGs

close to the metaphosphate composition [24, 27]. Despite this, there are subtle, but significant,

differences between spectra from samples of varying gallium content. The significant change

in the FTIR spectra is the reduction in the intensity of the band at 1150 cm−1 with increasing

gallium content. This band arises from modes involving Q1 end groups associated with Na+

ions [25] and perhaps it is not surprising that its intensity should be reduced as the Na+ ions are

replaced by Ga3+ ions. However, in agreement with the results from the 31P NMR study, this

21

change illustrates that the bonding interaction of the Ga3+ ions with the Q1 end groups is very

different to that of the Na+ ions. The other clear change in the infrared spectra is the gradual

change in the shape of the two νs(P–O–P) bands from two overlapping peaks in the spectrum

from the sample containing no gallium to one broad feature in that from the sample with the

highest concentration of gallium (5 mol% Ga2O3). It is common for the infrared spectra from

CaO-Na2O-P2O5 glasses to exhibit a split νs(P–O–P) absorption band [28], and it is known that

the energy of the νs(P–O–P) mode varies with the P–O–P bond angle because this angle

affects the amount of π bond character of the bridging bonds and hence their vibrational force

constants [26]. In the infrared spectra of Ga2O3-P2O5 glasses, only one νs(P–O–P) band is

observed at 760 cm−1 [26]. Hence, it is probable that the gradual change in the shape of the

νs(P–O–P) band with gallium content in the spectra presented here is caused by the growth of

a peak at 760 cm−1 between the partially overlapping peaks at 770 and 720 cm−1. Again this

change provides evidence for a bonding interaction between the phosphate chains and the

Ga3+ ions. The final and smallest change is the growth of a shoulder observed in the infrared

spectra from the samples with the highest gallium content at ~610 cm−1 on the on the high

frequency side of the broad peak attributed to O−P−O deformation modes . Although this

feature is only very weak, even in the spectrum from the sample containing 5 mol% Ga2O3, its

appearance provides some evidence for the presence of GaO4 tetrahedra in the glass structure.

This result does not contradict the 71Ga NMR result since the vibrations associated with GaO6

octahedra are expected to lie under the broad absorption due to the O−P−O deformation

modes [29] and are not observable here. Both the FTIR and 71Ga NMR spectra are consistent

with most of the gallium present in octahedral coordination with a small proportion

tetrahedrally coordinated.

The Raman spectra, however, shows one significant change, which is the change in shape of

the feature due to O−P−O deformation modes from two overlapping peaks at 380 and 340

22

cm−1 in the spectrum from the sample containing no gallium to one broad peak centred at 350

cm−1 in that from the sample containing 5 mol% Ga2O3. A peak at 350 cm−1 has previously

been observed in the Raman spectra from Ga2O3-P2O5 glasses [26]. The intensity of this peak

grew as a function of gallium content and consequently was assigned to symmetric bending

vibrations of P−O−Ga linkages. The changes in the Raman spectra observed here are

consistent with the growth of a band at 350 cm−1 with increasing gallium content and thus

provide further evidence for P−O−Ga bonding interactions. Furthermore, since the previous

vibrational study on Ga2O3-P2O5 glasses concluded that the nature of the P−O−Ga bonding

was highly covalent, it is likely that the P−O−Ga bonding in the glasses studied here has the

same character.

Thus, the vibrational spectroscopy provides evidence of the covalent nature of the P−O−Ga

interaction. This observation is consistent with the measured degradation rates which showed

a decrease with increasing gallium content, i.e. the Ga2O3-doped PBGs are highly

polymerised with the gallium increasing the covalency of the bonding and leading to a more

durable glass.

3.Conclusion

This paper reports the production of a novel quaternary Ga2O3-doped PBGs (0, 1, 3, and 5

mol% Ga2O3) with their antibacterial properties, physico-thermal properties, solubility, pH

change and ion release. The data obtained from the thermal and solubility analyses were

attributed to the packing density of the 45mol% P2O5 compositions. The solubility was seen to

decrease with increasing Ga2O3 mol%; FTIR and Raman spectroscopy provide some insight

into the likely reason for this via an understanding of the network bonding characteristics.

Both 0 and 1 mol% Ga2O3 compositions showed a gradual increase in pH with time, and this

was due to the depletion of H+ in the solution occurred resulted from its substitution with that

of Na+ and Ca2+ ions released into the solution . The ion release profiles exhibited similar

23

trends to the degradation rates obtained, and a decrease in the rate of release was seen for the

P ions with increasing Ga2O3 content. It was suggested that these ions were branched and

cross-linked with the Ca ions.

In disk diffusion assays, these compositions demonstrated antibacterial effects predominantly

against Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli , along with

small effect on methicillin-resistant Staphylococcus aureus and Clostridium difficile. Overall,

1 mol % Ga2O3-doped PBGs investigated was sufficient to mount a potent antibacterial effect

against the test organisms, and these compositions also gave excellent long-term release of

Ga3+ ions into the medium. Our findings suggested that this Ga2O3 containing glasses, 1

mol% in particular, holds promise as an antimicrobial agent and could offer some advantages

over conventional therapeutic agents as antibiotic-resistant organisms (even those with multi-

drug resistance) are likely to be sensitive to Ga3+. This is likely due to the fact that Ga3+ works

by a completely different mechanism to conventional drugs. Moreover, the fact that Fe3+

levels are so low in human tissues, and that Ga’s activity is increased when Fe3+ is limited [4],

raises the possibility that Ga3+ may be more effective in vivo than our in vitro data indicates.

These data, along with the fact that Ga3+ is FDA approved (for i.v. administration) and there

is a dearth of new antibiotics in development, make Ga3+ a potentially promising new

therapeutic agent for bacterial pathogens especially MRSA and C. difficile. Moreover, Ga3+ is

reported to inhibit bone resorption and stimulate bone formation through its action on

osteoblasts [4] and hence the Ga2O3 doped novel glass composition reported in this study

might well have applications in bone tissue engineering.

4. Experimental

4.1.Preparation of gallium-doped phosphate-based glasses

Phosphate-based glasses were produced using NaH2PO4 (BDH), P2O5 (Sigma), and CaCO3

(BDH). For the production of gallium containing PBGs, Ga2O3 (Sigma) was also used as

24

shown in Table I. The required amount of chemicals were weighed and placed into a

Pt/10%Rh crucible (Johnson Matthey, Royston, UK). The crucible was then placed in a

preheated furnace at 1100°C for 1hour. The molten glass was then poured into graphite

moulds, which had been preheated to 350°C. The glass samples were allowed to cool to room

temperature, and the resulting glass rods were cut into discs by using a rotary diamond saw

(Testbourne Ltd., Basingstoke, UK). Density measurements were conducted on triplicate

samples using Archimedes’ Principle.

4.2.Degradation study

Ga2O3-doped PBGs rods (5 mm diameter and 2 mm thickness) with different contents of

gallium ions were placed in plastic containers, filled with 50 ml of deionised water (pH 7±0.5),

and then placed in a 37°C incubator. At various time points (6, 24, 48, 72 and 120h), the

three disks were taken out of their respective containers, and excess moisture was removed by

blotting the samples dry with tissue prior to weighing them. All the disks were placed into a

fresh solution of deionised water and placed back into the 37°C incubator. To obtain the rate

of weight loss, the initial weight (M0) of each sample was measured as well as the weight at

time t (Mt) to give a weight loss per unit area thus: weight loss=(M0–Mt)/A, where A is the

surface area (mm2). The measurements were carried out in triplicate, and the weight loss per

unit area were plotted against time. The slope of this graph gave a degradation rate value in

terms of mg.mm-2 h-1, which was determined by fitting a straight line of the form y = mx

through the origin.

4.3. pH measurements

The pH measurements of the degrading medium were taken at each time point (6, 24, 48, 72

and 120h) using a Hanna Instruments pH 211 Microprocessor pH meter (BDH, UK) with an

attached glass combination pH electrode (BDH, UK). The pH electrode was calibrated using

pH calibration standards (Colourkey Buffer Solutions, BDH, UK).

25

Both dissolution studies and standards, for ion release study, were prepared using high purity

water. This was obtained from a PURELAB UHQ-PS system (Elga Labwater, UK), which

polished the water obtained from an existing water purification system, to a purity level of

18·2 MΩcm-1 resistivity.

4.4. Ion release study

Ion release studies were simultaneously conducted, and the medium was analysed for Na+ and

Ca2+ using ion chromatography (Dionex, UK). Both Gallium and Phosphorous ion release

was measured using inductively coupled plasma mass spectrometry, ICP-MS, which is an

analytical technique that determines the elements content in the samples. It is accomplished

by counting the number of ions at a specific mass of the element and detects only elemental

ions and can determine the individual isotopes of each element. The ICP-MS (Spectromass

2000 by SPECTRO) was used to determine amounts of both gallium and phosphorous ions

released from all tested glass compositions at the previously mentioned time points. The

instrument detection limit of the gallium and phosphorous is in the range 1-10 ppt. However,

instrument was calibrated for the predicted concentration in the range 0.1-1000ppb by mixing

single element standards obtained from Sigma and diluted in ultra pure water.

4.5. Inhibition of microbial growth by gallium-doped PBGs

Ga2O3-doped PBGs (1, 3, and 5 mol % Ga2O3) were investigated for their ability to inhibit

microbial growth using a disk diffusion methodology (BSAC Disk Diffusion Method for

Antimicrobial Susceptibility Testing, Version 4, 2005). Isosensitest agar (Oxoid, Basingstoke,

UK) plates were inoculated with a standardized culture of S. aureus (NCTC 6571), E. coli

(NCTC 10418) and P. aeruginosa (PA01). Columbia agar (Oxoid, Basingstoke, UK) with 2%

NaCl plates were inoculated with MRSA-16. In the case of the anaerobic bacterium C.

difficile, Wilkins-Chalgren agar plates supplemented with 5% horse blood (E & O

Laboratories, UK) were used. Ga2O3-doped PBGs disks of 5 mm diameter and 2 mm

26

thickness were then placed on the inoculated plates. Disks not containing any gallium were

used as negative controls. These plates were then incubated overnight in air at 37°C except C.

difficile which was incubated for 48 hours in an anaerobic chamber. The diameters of any

zones that had formed around the disks were measured in triplicate using calipers.

P. aeruginosa (PA01) which showed the highest susceptibility to the Ga2O3-doped PBGs

were inoculated into 10mL of nutrient broth and incubated overnight at 370C with 200 rpm

agitation in an Orbital Shaker (Stuart Scientific, UK). The overnight cultures were used to

inoculate 5mL volume of phosphate buffer saline (PBS; Oxoid) to a standardized optical

density of 0.03 at a wavelength of 600 nm (OD600). Ga2O3-doped PBGs disks of 5 mm

diameter and 2 mm thickness were added to each tube, with the gallium free disk (0 mol%

Ga2O3) used as controls. The tubes were then incubated at 370C. At various time intervals (1,

12 and 24 h) serial dilutions of the suspensions were carried out in PBS. 50 µl volumes of the

suspension and each dilution were spread onto McConkey agar (Oxoid, Basingstoke, UK)

plates. The plates were then incubated aerobically at 30°C for 48 h. For each type of disc,

viable counts (colony forming units; CFUs) were conducted in triplicate.

4.6. Thermal and Structural analysis of the gallium-doped phosphate-based glasses

4.6.1. Thermal Analysis

A portion of each glass sample was crushed into powder using a vibratory agate mill, and the

glass transition temperature (Tg) was determined using a Pyris Diamond Differential Scanning

Calorimetry (Perkin-Elmer Instruments, UK). The instrument was calibrated using the

manufacturer’s instructions, with indium and zinc as standards, and all tests were carried out

under nitrogen purge. Samples (n=3) of 5mg were heated, cooled and reheated from 25 to

550 °C at 100°C.min-1. Tg was calculated by the onset of change in the endothermic direction

(upwards) of the heat flow.

27

4.6.2.NMR

23Na MAS NMR experiments were conducted using a 3.2 mm diameter rotor spinning at 30

kHz. Spectra were acquired using a Bruker Avance II spectrometer attached to a 14.1 T

magnet (23Na Larmor frequency 158.7 MHz). Aqueous NaCl was used as a reference, with

the sharp resonance from this set to 0 ppm. The liquid 90° pulse length was determined as 2.5

µs, although a much shorter pulse length (0.5 µs) was used on the solid samples. A one-pulse

sequence was used, with a recycle delay of 5 seconds. Certain 23Na spectra were also

recorded at 7.05 T under similar conditions.

31P MAS NMR experiments were conducted using a 4 mm diameter rotor spinning at 10 –

12.5 kHz. Spectra were acquired using a Chemagnetics Infinity Plus spectrometer attached to

a 7.05 T magnet (31P Larmor frequency 121.5 MHz). NH4H2PO4 was used as a secondary

reference compound, the signal from this set to 0.9 ppm. A pulse length of 1.5 µs was used

(corresponding to a ~30° tip angle), with a recycle delay of 5 seconds.

71Ga MAS NMR experiments were conducted at 14.1 T (71Ga Larmor frequency 183.0 MHz)

using a Bruker Avance II spectrometer and a 3.2 mm rotor, spinning at approximately 18 kHz.

A one-pulse sequence was used with a pulse length of 0.75 µs corresponding to a tip angle of

~30°, and a recycle delay of 2 seconds. Spectra were references to aqueous Ga(H2O)63+ at 0

ppm. The 5 molar % Ga2O3 sample was also investigated at 18.8 T using a 2.5 mm probe

spinning at 22 kHz and similar experimental parameters.

All spectra were processed using TOPSPIN 2.0 or Spinsight and fitted using either dmfit2007

[29] or QuadFit [30].

4.6.3.Vibrational Spectroscopy

Infrared spectra were recorded in transmission mode on a Biorad FTS175C spectrometer

controlled by Win-IR software. Samples were diluted in dry KBr and scanned in the range

4000-400 cm−1. Each spectrum was the result of summing 64 scans.

28

The Raman data were collected using a Jobin Yvon microRaman module attached to an

Olympus microscope (BX40). The illuminating laser line was at 632.8 nm (HeNe). The

spectrometer had a 1200 gr/mm grating. The spectra were recorded on a liquid nitrogen

cooled CCD. Typical sample exposure times were 10x10 secs.

Acknowledgements

This work was supported by the EPSRC, UK grant no. GR/T21080/01, EP/C000714/1 and

EP/C000633/1. The authors would like to thank Nick Foster for his help with the collection of

the Raman data. This work was undertaken at UCLH/UCL which received a proportion of

funding from the department of health’s NIHR Biomedical Research Centres Funding

Scheme, UK.

29

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