8/11/2019 Elucidating the Antimicrobial Mechanisms of Silver: Significance of Higher Oxidation States and Reduction Potential

1/1

y =6.0638x+10195

R =0.9164

0

5000

10000

15000

20000

25000

30000

35000

0 500 1000 1500 2000 2500 3000

1/[Trp](M-1)

Time (seconds)

Elucidating the Antimicrobial Mech

Significance of Higher Oxidation St

CSC 2012, Calgary, Alberta

David Lischuk B.Sc, P. Chem; Carla Spina, Ph.D

Exciton Technologies Inc.

Abstract.

For centuries, silver has been used in medicinal and sanitation applications. As early as the 8th century, the

use of silver as a blood purifier and for bad breath was documented by the Muslim physician Avicenna1. The

biocidal property of silver and other metals has bee n described as an oligodynamic effect. From the Greek

(oligos = few, and dynamis = power), it is a term used to describe how low metal ion concentrations exert

powerful biocidal effects toward lower life forms, yet are non-toxic to humans and other higher life forms,

with the relative toxicity as described in the Horsfall Series:

Ag > Hg > Cu > Cd > Cr > Ni > Co > Zn > Fe > Ca

However, the exact biochemical mechanisms or actions and interactions of silver are still under debate, where

a hypothetical sub-division of the series may be: Ag3+> Ag2+> Ag+

To elucidate the impact of silver oxidation state and reduction potential, the biological chemistry of silver

compounds and select amino acids are explored as a model system. In particular: kinetic NMR and UV-Visible

spectrometry experiments will explore the biological oxidative effects of Ag7NO11, AgO, Ag2O, and AgNO3in

aqueous medium.

Introduction.

Silver has been a ubiquitous antimicrobial agent from early history to contemporary wound care. Since the

first silver wound care device brought to market in 19894 , there has been an surge of silver-based wound care

devices available, containing a variety of silver species as seen in Table 1.

Although it is well-accepted in the wound care community that silver must be present in an ionic state to

provide antimicrobial activity, with the vast array of medicinal silvers available and the risks of antimicrobial

resistance, a more thorough understanding of the mechanism of action of silver is needed6. Current literature

has identified three primary mechanisms: protein complexation and denaturation7; DNA complexation and/or

intercalation, impeding transcription8; and cellular respiration interference, either as an electron sink or via

disruption of membrane potentials9. This work examines the effect of medicinally relevant silver compounds,

of various reduction potentials and oxidation states, on amino acid oxidation as a model system for protein

degradation. The work presented here demonstrates the unique activity of various silver compounds and

supports further investigation into oxidative mechanisms towards antimicrobial activity.

Methods.

UV-Visible spectra were obtained on a Hewlett Packard 8452A UV-Visible spectrophotometer, using UV-rated

polymeric cuvettes (path length = 10 mm) unless otherwise stated. Tryptophan, glycine and D2O were

obtained from Sigma Aldrich, silver(I,III) oxide and silver(I) oxide were obtained from Alfa Aesar, and silver

nitrate was obtained from Anachemia; all chemicals were used without further purification. Ag7NO11was

prepared using published procedures5and stored protected from light 4 oC until needed. Water for

spectrophotometry was generated using a custom Reverse Osmosis and Deionizer system (Claysmore Spring

Water).

A UV-Vis calibration curve for Tryptophan (Trp) was generated using max

= 279 nm peak, with a linear range of

0.020.30 mM (Figure 1). Glycine (Gly) exhibited weak UV-Vis absorbance (Figure 2), therefore was studied

via 1H NMR spectroscopy, as detailed below. Preliminary UV-Vis degradation profiles for silver-Trp were carried

out in a syringe, filtering insoluble components with a 0.2 m syringe filter. A typical procedure went as

follows: 9.0 ml Trp solution (0.04 g/L, 0.20 mM) was loaded into a syringe with a clinically relevant

concentration of the identified silver compound (1 mg Ag equivalent/mL solution). The reaction mixture was

agitated for a specified time, then filtered through a 0.2 m syringe filter directly into a cuvette, acquiring a

UV-Vis spectrum (190-820 nm). Blank solutions were prepared using the equivalent volumes and

concentrations of silver without Trp; blank absorbance recorded at 279 nm . From each reaction syringe, 3

spectra at different time points were obtained for the TrpAg system up to 250 minutes. For those

preliminary systems demonstrating reactivity, subsequent kinetic studies were undertaken using a bulk

reaction sampling procedure. Briefly, 200 mL of stock Trp solution was prepared, to which 0.25 g Ag-OXS was

added under vigorous stirring. 2 ml samples were removed every 5 minutes for a period of 2 hours, filtered,

and spectrum obtained.

Proton NMR studies were performed at the NMR laboratory at the University of Alberta; an Agilent/Varian

Inova 400 MHz instrument was used for all spectra. A concentrated stock solution of Gly in D2O (100 g/L, 1.3

M) was prepared and sealed in a glass ampoule until needed. The concentrated stock solution was used to

ensure that spectra could be obtained rapidly, in order to obtain reliable kinetic data. A baseline spectrum was

obtained, and then 10 mg of either Ag7NO11or Ag2O was added directly to the tube, reacted for 30 minutes

until the first spectrum was obtained. Consecutive spectra were obtained at 30 minute intervals until 3 hours

had elapsed. The procedure was repeated for the other oxide, in fresh solution.

Acknowledgements.

The authorswouldlike tothank Dr.Michael Serpe for the use ofhisspectrophotometer,Mr.Avijeet Sarkerand Mr.Mark Mizolskifor helpfuldiscussions,and the University ofAlberta NMR laboratory for the performance ofthe NMRexperiments.

References.

1)Bhattacarya R, Mukherjee P.AdvancedDrugDelivery.Reviews. 2006; 60: 1289-1306. 2)Wadhera A,Fung M. DermatologyOnline Journal.2005; 11: 12 3) Fox, CL.ArchSurg .1968; 96(2): 184-188. 4)http://www.accessdata.fda.gov/scripts/cdrh/c

Trauma.1987; 27(3): 301-303.

7)Liau SL,et al. Lettersin AppliedMicrobiology. 1997; 25: 279-283. 8)Modak SM,Fox CL. BiochemistryPharmacology.1973; 22(2391): 404.9) Dibrov P,et al.AntimicrobialAgents inChemotherapy.2002; 46(8): 2668-2670. 10)Harriman,A. J PhysCh

ChemistryandPhysics: 88thEdition(Chemical Rubber Company),2007

This work was sponsored by Exciton Technologies Inc.

SilverSpeciesChemical

Compound

Oxidation

State

Solubilityin

water

(maximum

ppm Ag)

Generation ofclinically

relevant silverspecies

Metallic Silver Ag Ag0 0.0

Oxidation (byairor

biological fluids):

generatingsurfaceAg2O

Silver

Sulfadiazine

(SSD)

Ag(C10H9

N4O2S)Ag1+ 0.3

Dissolution: generating

Ag1+and the antibiotic

sulfadiazine

SilverChloride AgCl Ag1+ 1.4

Dissolution: generating

Ag1+and the Cl -counter-

ion

Silver(I)Oxide Ag2O Ag1+ 22.4

Dissolution: generating

Ag1+andOH-via reaction

with water

SilverSulfate Ag2SO4 Ag1+ 1638

Dissolution: generating

Ag1+andthe SO4-

counter-ion

SilverNitrate AgNO3 Ag1+ 775000

Dissolution: generating

Ag1+and the NO 3-

counter-ion

SilverSodium

Hydrogen

Zirconium

Phosphate

Ag0.1-0.5)Na(0.1-0.8)H(0.1-0.8)Zr2(PO)3

Ag1+ n/a

Dissociation/ion

exchange: generatingfree

Ag1+which canbe

replacedin thespeciesby

anequivalent cation from

the wound fluid

SilverOxysalts Ag7NO11Ag1+, Ag2+,

Ag3+n/a

Decomposition ofthe

solidmaterial: generating

Ag1+, Ag2+, Ag3+, aswell

asO 2, NO3-, and other

species

Table 1: Propertiesof Clinically Common Silver Species

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

255 355 455 555 655 755

Absorbance

(nm)

198 g/LGly

40 g/LGly

Figure 2: UV-Visspectraof glycine at 198 and 40 g/L.

0

0.5

1

1.5

2

2.5

3

180 280 380 480 580 680 780

Absorbance

(nm)

y =5.1254x+0.0204

R =0.9993

0

0.5

1

1.5

2

2.5

0 0.1 0.2 0.3 0.4 0.5

Absorbance

[Trp] (mM)

Figure 1: UV-Visspectraof tryptophanat aseries of applicable concentrations.Inset:Calibration curve, using the 279 nm peak.Note that the red point in the inset isnotincluded in the curve, indicating the limit of linearity.

0

0.2

0.4

0.6

0.8

1

1.2

190 290 390 490 590 690 790

Absorbance

(nm)

a

0

0.2

0.4

0.6

0.8

1

1.2

190 290 390 490 590 690 790

Absorbance

(nm)

b

0

0.2

0.4

0.6

0.8

1

1.2

190 290 390 490 590 690 790

Absorbance

(nm)

c

0

0.2

0.4

0.6

0.8

1

1.2

190 290 390 490 590 690 790

Absorbance

(nm)

d

Figure 3: Typical spectraof tryptophanupon contact with varioussilver species.Clockwise from upper-left: a)Ag-OXS; b)AgO; c) Ag2O; d)AgNO3.The initial spectrumin all casescomesfrom the stocktrp solution.

y =7.0452ln(x)+48.864

R =0.9788

0

10

20

30

40

50

60

70

80

90

100

0 50 100 150 200 250

%

[Trp]

Time (min)

Figure 4: %change in [Trp] over time, upon exposure to Ag-OXS

Figure 5: Linear fit of 1/[ Trp]vs.time, showing an approximately linear relation

Figure 6: Reference 1HNMR spectraof a)glycine stocksolution (1.3 M in D 2O); b)glycine and Ag2O; c)glycine and Ag7NO11

*Solubility in 0.1 M NaNO3,Clinical Chemistry, 199516

Information not available in published literatureSolubility not applicable asAg 7NO11slowly decomposesin solution (dataon file)