The University of QueenslandThe Petroleum and Petrochemical College Chulalongkorn University

Presented by Philip Martin BarberSchool of medicine, The University of Queensland

A NOVEL SILVER HYDROGEL DRESSING FOR BURN WOUNDS

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Patients with serious burns require specialized treatment minimizes trauma and mortality

Well-known pathogens that infect burns and are resistant to broad spectrum of antimicrobial agents are

Burn wounds and infection

Methicillin-resistant Staphylococcus aureus (MRSA)

Pseudomonas aeruginosa

Burn damage Infection damage

Microbial load ≥ 105 CFU/g of tissue

infectious

http://www.worldwidewounds.comhttp://www.cdemcurriculum.org

http://www.pitchcare.comhttp://www.medindia.net

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Characteristic

Is a polymeric material Does not dissolve in water Swells considerably in an aqueous medium

Absorbs exudate from wounds

Maintains a moist environment

Has painless removal and leaves no residue

Is transparent and enables observation of the healing process

Advantages

Hydrogel as a wound dressings

http://www.hartmann.com.au

http://www.ec21.com

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• Hydrophilicity

• High swelling capacity

• Thermal stability

• Lack of toxicity

Unique properties

AMPS hydrogel

• Water treatment

• Construction chemicals

• Hydrogels for medical purposes

2-Acrylamido-2-methylpropane sulfonic acid (AMPS)

Applications

AMPS AMPS-Na+

Polymer Monomer

Silver as an antimicrobial agent

Silver Among the metals with antimicrobial properties, silver has the most effective antibacterial actionand the least toxicity to animal cells

History of silver Silver was commonly used in medical treatment such as those wounded soldiers in World War I

Discovery of antibiotics Once antibiotics were discovered, the use of silver decreased

Increasing antibiotic resistance There has recently been a renewed interest in using silver as antimicrobial agents

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Inhibitory action of silver

Protein Inhibition: Ag binds to thiol group of enzymes (-SH) S-Ag

Destruction of cell membranes: Ag binds to cell walls and outer cell membranes altering their function

Inhibition of DNA replication: Ag intercalates between the purine and pyrimidine base pairs

Denaturing DNA: Ag disrupts hydrogen bonding between two anti-parallel strands

www.burnsurgery.org

Novel dressing combined hydrogel & silver

Hydrogels have been used on burns to keep them moist & stimulate healing

Silver containing dressing products have been used in burn care to fight infection

Novel silver hydrogel

New Idea

Promote would healing Antimicrobial activity

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Synthesis Characterization

The University of QueenslandThe Petroleum and Petrochemical College Chulalongkorn University

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0.1 mol % MBA AgNO3

100 ml of 40% AMPS-Na+ solution pH 7.0

Transfer to nylon bags

Stir overnight Stir 30 min

Synthesis of novel silver hydrogel

1-10 mM (0.17-1.7 g/l)

Gamma irradation(25 kGy)

polymerization of AMPS polymer in a present of MBA crosslinker and a reduction of silver ion to form silver nanoparticles (SNP) infused in the crosslinked polymeric network in the field of gamma irradiation

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Silver nanoparticles formation

The appearance of the radiated hydrogel pads darkened with increasing silver concentration

neat hydrogel 2.5 mM silver 5 mM silver 10 mM silver

neat hydrogel 5 mM silver 10 mM silver

SEM images revealed that silver hydrogels hadrough surfaces with particles on the surfaces

2.5 mM silver 5 mM silver 10 mM silver

The TEM images indicated that the size of the SNPs were less than 25 nm

The average particle sizes were 8.08, 6.53, 4.83 nm for 2.5, 5 and 10 mM silver hydrogel, respectively

Evaporative water loss of normal skin and each degree of burn skin (Nilsson, G.E., 1997)

Evaporative water loss (g/h.m2)Healthy skin 8.5 ± 0.5First degree burn 11.6 ± 1.1Second degree burn 178.1 ± 5.5Third degree burn 143.2 ± 4.5

maintains moist environment of burn wounds

Advantage to be use as a

wound dressing

Water vapor transmission rate (WVTR)

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Conc of silver

hydrogel (mM)

WVTR

(g/m2/h)

neat 97.5 ± 5.0

2.5 102.0 ± 4.2

5 95.2 ± 4.9

10 86.7 ± 3.1

Hydrogel discs were used to cover the top of bottles All hydrogels have WVTR values < evaporative water

loss of 2nd and 3rd degree burns

WVTR is a measure of the passage of water vapor through a substance (http://en.wikipedia.org/)

Two stages of release1. Initial rapid release within the first 72 h up to 70%2. Continuous slight release from 72 h-240 h 80-90%

Silver hydrogel potential silver release dressing on burns for 72 h

Cumulative silver release

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Silver hydrogels disc was immersed in SBF (simulated body fluid) solution. At each time point, the immersion solution was collected to measure the silver content using atomic absorption microscopy

None of the hydrogel were toxic to cells relative viability was greater than 90 %

Pretest for selection of the best dressing

P. aeruginosa: Gram (-)

MRSA: Gram (+)

Extraction media of hydrogels were incubated with cells and MTT cytotoxicity assay was performed

**5 mM silver hydrogel has the lowest silver content that can effectively inhibit the tested bacteria it was selected to be the best dressing

Hydrogels were incubated with bacterial innocula and at time intervals, aliquots were sampled and the colony count were performed

5 and 10 mM silver hydrogel can inhibit P.aeruginosa growth within 3 h and 12 h for MRSA

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Intensive Cytotoxicity Assessment

The University of QueenslandThe Petroleum and Petrochemical College Chulalongkorn University

To examine the cytotoxicity of the selected novel silver hydrogel dressing in comparison with some commercially available silver products

To compare the efficacy and sensitivity of 4 different cytotoxicity assays

- Surviving cell count - MTT

- CellTiter-Blue® - Photovisualization (Toluidine Blue staining)

To investigate the cytotoxicity of silver agents on two different cell lines

- HaCaT (keratinocytes, immortal cell line) - NHF (normal human fibroblasts, primary cell line)

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Objectives

ActicoatTM PolyMem

Silver® FlamazineTM Neat hydrogel Silver hydrogel

(5 MM AgNO3)

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Experimental design

Control ActicoatTM PolyMem Silver®

FlamazineTM cream Neat hydrogel Silver hydrogel

Cells

Insert

CreamDressing

The silver agents were individually placed on top of a Nunc polycarbonate insert and were incubated with the cell cultures for 24, 48 and 72h

1 Surviving cell count trypsinize cells then count cell no. Haemocytometer

2 MTT (colorimetric assay) mitochondrial metabolism Microplate reader (570)

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1. Four cytotoxicity assays used

Relative cell viability (%) of HaCaT at 24 h exposure

3 CellTiter-Blue (fluorometric assay) metabolic capacity Microplate reader (ex 544 nm, em 590 nm)

4 Photovisualization Stain cells with Toluidine Image Pro Plus v5.1 software: calculate surviving area

Mitochondrialreductase

Untreated control Treated sampleMTT MTS

http://www.promega.com

1. Comparison of four cytotoxicity assays

MTT provided closest value to the average values from all. 1. comparatively low SD 2. rapid protocol 3. relatively low cost

But MTT measures metabolic activity not the viable cells surviving cell count was performed (confirm viable cells)

Acticoat & Flamazine Toxic to HaCaT (24 h)

* p < 0.01

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Cytotoxicity of burn wound products on HaCaTs (24 h)

Control Acticoat

PolyMem Silver Flamazine

Neat hydrogel Silver hydrogel

2. Cytotoxicity on Cells up to 72h

MTT assay of HaCatexposed to burn wound products

5060708090

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24 h

48 h

72 h

*

*

*

** *

*

* **

Rela

tive c

ell v

iab

ilit

y (

%)

MTT assay of NHFexposed to burn wound products

Contro

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Actico

at

PolyMem

Silv

er

Flam

azin

e

Neat h

ydro

gel

Silver

hyd

roge

l5060708090

100110120130140150

24 h

48 h

72 h

** * *

*

Rela

tive c

ell v

iab

ilit

y (

%)

Surviving cell count of HaCatexposed to burn wound products

5060708090

100110120130140150 24 h

48 h

72 h

* ***

*

*

*

*

**

*

Rela

tive c

ell v

iab

ilit

y (

%)

Surviving cell count of NHFexposed to burn wound products

5060708090

100110120130140150 24 h

48 h

72 h

* * **

Rela

tive c

ell v

iab

ilit

y (

%)

A C

B D

72 h exposure

HaCaT NHF

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Control

Acticoat

Silver hydrogel

HaCaTs treted with Acticoat & Flamazine increase ability to survive at 72 h

MTT assay of HaCaT MTT assay of NHF

Surviving cell count of HaCaT Surviving cell count of NHF

Novel silver hydrogel showed no cytotoxicity to both cell lines

dense cells

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Antimicrobial efficacy

The University of QueenslandThe Petroleum and Petrochemical College Chulalongkorn University

To examine the antimicrobial efficacy of the novel silver hydrogel dressing and silver hydrogel* kept at RT for 1 year in comparison with two common silver dressings (neat hydrogel is negative control)

To compare the efficacy and sensitivity of 3 different antimicrobial assays- Disc diffusion method - Microbicidal measurement (Plate count method)- Live/Dead® BaclightTM bacterial viability kit

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Objectives

1. Disc diffusion method

PolyMem Silver® & neat hydrogel no zone of inhibition for most tested microbes.

Silver hydrogel & 1 year old silver hydrogel* inhibit microbial growth only under the dressings

ActicoatTM the most effective antimicrobial activity (13.9-18.4 mm).

Only ActicoatTM treatment inhibition zone for MRSA.

Inhibition Zone length in diameter (mm) Micro-orgamism ActicoatTM

PolyMem

Silver®

neat

hydrogel

silver

hydrogel

silver

hydrogel*

P. aeruginosa 16.8 ± 1.0 0 3.8 ± 0.8 10.2 ± 0.2 10.3 ± 0.1

MRSA 13.9 ± 0.3 0 0 0 0

MSSA 16.3 ± 0.5 10 ± 0.0 0 10.7 ± 0.9 11.8 ± 1.3

A. baumanii 16.3 ± 0.5 0 0.5 ± 0.8 10.0 ± 0.0 10.0 ± 0.0

VRE 16.0 ± 0.3 0 0 9.6 ± 0.3 9.5 ± 0.7

C. albicans 18.4 ± 0.5 9.8 ± 0.5 2.5 ± 3.0 9.0 ± 1.7 9.1 ± 1.3

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The bacteria inocula were spread on Tryptic Soy Agar plates and the hydrogel discs were placed on the agar

2. Plate count method

ActicoatTM inhibit microbial growths in 4 h.

PolyMem Silver® poorest action

Silver hydrogel inhibit microbial growths in 24 h, only VRE: not completely inhibited

Silver hydrogel* inhibits P. aeruginosa and C. albicans (no inhibition in antibiotic resistant strains)

Oxoid nutrient broth

Hydrogels were incubated with bacterial inocula and at time intervals, aliquots were sampled and the colony count were performed

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3. Live/Dead® BaclightTM kit

green flu (stains live and dead cells)red flu (stains only dead cells).

ActicoatTM inhibit microbial growths 99% within 4 h

PolyMem Silver® poorest action, in VRE promote growth up to 132.73 %

Silver hydrogel inhibited microbial growth by 95-99% at 24 h.

Silver hydrogel* inhibited only MSSA growth 24

Hydrogels were incubated with bacterial inocula and at time intervals, aliquots were sampled and were mixed with the commercial reagents

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PolyMem Silver® low cytotoxicity & poorest antimicrobial action

Silver hydrogel least cytotoxicity & good antimicrobial action (24 h)

*** High Ag content high cytotoxicity VS high antimicrobial action

★ Balance of Cytotoxicity & Antimicrobial action

Comparison of Efficacy

ActicoatTM best antimicrobial action (4 h) & most cytotoxicity to human cells

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Silver hydrogel No cytotoxicity & Good antimicrobial activity Single step synthesis to form a ready to use sterile dressing

Relatively economical to produce

★ Competitive silver dressing for burn care in the future

Conclusion

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Dr. Benjawan Boonkaew Prof. Pitt Supaphol Dr. Leila Cuttle Margit Kempf Prof. Prasit Pavasant Assoc. Sirirat Rengpipat

Acknowledgements

References Hydrogels containing silver nanoparticles for burn wounds show antimicrobial activity without cytotoxicity. Journal of

Applied Polymer Science (2014) DOI: 10.1002/app.40215

Antimicrobial efficacy of a novel silver hydrogel dressing compared to two common silver burn wound dressings: ActicoatTM and PolyMem Silver. Burns (2014) 40, 89.

Cytotoxicity testing of silver-containing burn treatments using primary and immortal skin cells. Article in press Burns (2014) doi.org/10.1016/j.burns.2014.02.009