Adsorption, system release and antimicrobial properties of chlorhexidine on nanohydroxyapatite...

ADSORPTION, SYSTEM RELEASE AND ANTI-MICROBIAL PROPERTIES OF CHLORHEXIDINE ON NANOHYDROXYAPATITE COATINGS

J Barros, L Grenho, CM Manuel, OC Nunes,

LF Melo, FJ Monteiro, MP Ferraz

[email protected]

INEB – Instituto de Engenharia Biomédica

Universidade do Porto

Porto, Portugal

INEB - Instituto de Engenharia Biomédica

LEPAE – Laboratory for process, environmental and energy engineering Universidade do Porto, Porto, Portugal

Nanohydroxyapatite

Biocompatibility Bioactivity

Osteocondutivity Osteoinduction

Ferraz et al, (2004). Journal of Applied Biomaterials & Biomechanics

Antimicrobial activity

Frequency of main pathogenic species among

orthopedic clinical isolates of implants-

associated infections.

Darouise. (2001). Clinical Infections Diseases

Campoccia et al. (2006). Biomaterials

Devices-associated infections

Biofilm – is a structured community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface.

Biofilm formation

Manuel. 2007. Thesis doctoral.

Chlorhexidine digluconate (CHX)

Anti-microbial activity:

• Gram negative bacteria

• Gram positive bacteria

• Fungi

Souza et al. (2011). Colloids and Surfaces B: Biointerfaces.

Acting Mechanism

Souza et al. (2011). Colloids and Surfaces B: Biointerfaces.

To study the adsorption of chlorhexidine digluconate (CHX) to

nanohydroxyapatite discs sintered at 830˚C (nanoHA830)

To study the release system from these coatings

To evaluate the in vitro anti-adhesive and anti-bacterial

activity of CHX + nanoHA coatings

Purposes

Materials and methods

150 mg of nanoHA powder

1- Preparation of nanohydroxyapatite discs heat-treated at 830˚C

Sintering at 830˚C

Pressing at 2 MPa

nanoHA discs heat-treated at 830˚C (nanoHA830)


1. Adsorption of CHX on nanoHA830

Different solutions of CHX (5-1500mg/L) were prepared by

diluting an aqueous stock solution of 20% chlorhexidine

digluconate in sterile deionized water.

Adsorption experiments were carried out in a

batch system using nanoHA sintered at 830˚C

discs. The nanoHA830 discs were placed in 6-well

polystyrene microtiter plates and incubated with

each CHX concentration prepared previously and

moderately shaken for 24 h at 37˚C.

5 50 100 500 1500

UV–vis spectrophotometry, 254 nm wavelength


2. System release

1500 mg/L CHX + nanoHA830 Sampling time (h)

1, 24, 48, 72, 120, 168, 240 and 312

24 to 24 h

Fresh 0.9%NaCl

UV–vis spectrophotometry, 254 nm wavelength

Materials and methods 3. Minimum inhibitory concentration

Minimum inhibitory concentration (MIC) of CHX were determined for S. aureus ATCC 25923 by

micro-dilution method, according to what was defined by Clinical and Laboratory Standards

Institute. The MIC is the lowest concentration of a drug that prevents growth of a particular

pathogen.

Colune

1 Muller-Hinton Broth (200 µL)

2 Sterile water (200 µL)

4 Bacterial suspension (200 µL)

6-12 Chlorhexidine digluconate concentrations (20 µL) + Bacterial suspension (180 µL)

1- 150 mg/L CHX

Materials and methods 4. Antimicrobial studies

• Anti-adhesive properties • Anti-bacterial activity

Colony Forming Units

CFUs/mm2

CHX + nanoHA830

Pure nanoHA830

1,5×108/mL

24h/37°C

Batch system

120 rpm

Ultra-sonic batch

Bacterial detachment

Over time the bacteria/ml

was measured at 640 nm.

Suspension was placed in

96-well plates.

Results 1. Isotherm adsorption

Adsorbed CHX onto nanoHA discs exhibited Langmuir-type adsorption isotherm, with

a good correlation.

0,E+00

5,E-04

1,E-03

2,E-03

2,E-03

0 500 1000 1500 2000

Qe

(mg/

mm

2)

Ce (mg/L)

Langmuir model

2.6 × 10-3

1.5 × 10-3

1.0 × 10-3

5.0 × 10-4

Qe =

R2 = 0.9974

Ce

552.71Ce + 74915

Qe – Amount of CHX adsorved per unit area of nanoHA discs

Ce – Equilibrum CHX concentration, after incubation

Results 1.2 Materials characterization

Attenuated total reflectance (ATR) – FTIR.

0

20

40

60

80

100

120

4000 3700 3400 3100 2800 2500 2200 1900 1600 1300 1000 700 400

% t

ran

smit

ance

(a.

u)

Wavenumbers (cm-1)

nanoHA830

5 mg/L CHX+nanoHA830





*

*

•OH-

*PO4-3

*

*

*

*

•

•

Results 1.3 Materials characterization

5 mg/L CHX + nanoHA830

500 mg/L CHX + nanoHA830 100 mg/L CHX + nanoHA830 1500 mg/L CHX + nanoHA830

nanoHA830 50 mg/L CHX + nanoHA830

Results 2.System release

The 1500 mg/L CHX+nanoHA disc coatings showed a slow release over time for a period of several

days, being the amount of CHX released higher than MIC values for bacteria used.

0

4

8

12

16

20

24

28

0 50 100 150 200 250 300 350

Rel

ease

acc

um

ula

tive

co

nce

ntr

atio

n

(mg/

L)

Time (h)

MIC = 4 mg/L

1,00E+07

1,00E+08

1,00E+09

1,00E+10

3 24 48 72 120 144 168 172 196

Bac

teri

a d

ensi

ty (

bac

teri

a/m

L)

Time (h)

NanoHA830






1 × 107

1 × 1010

1 × 109

1 × 108

Results 3. Anti-bacterial activity

The CHX released from 1500 mg/L CHX + nanoHA and 500 mg/L CHX + nanoHA coatings inhibited the bacteria growth

during 9 days, being the amount bacteria/mL lower than initial bacteria density added.

•p 0.05, significant reduction compared to pure nanoHA830, according to Tukey HSD

*p> 0.05, no significant differences reduction compared to initial bacteria density added, according to Tukey HSD

*

• •

Initial bacteria density added

100%

70,6%

22,4%

0% 0% 0

10

20

30

40

50

60

70

80

90

100





Per

cen

tage

of

bac

teri

a ad

her

ed o

n s

urf

ace

s (%

)

Surfaces

Results 3. Anti-adhesive properties

During 24 hours of incubation were evaluated the anti-adhesive properties for 1500 mg/L, 500 mg/L,

100 mg/L and 5 mg/L CHX+nanoHA coating compared with pure nanoHA830 surface.

*

*p 0.05, significant bacteria adhesion compared to pure nanoHA830, according to Tukey HSD

Conclusions

Adsorbed CHX onto nanoHA discs exhibited Langmuir-type adsorption isotherms with

a good correlation (R2 = 0.9974).

The chlorhexidine digluconate was incorporated into the coatings via formation of a

chlorhexidine phosphate precipitate onto the nanoHA discs.

The morphological effect studied by SEM showed that CHX did not cause

morphological changes to the nanoHA surfaces.

The 1500 mg/L CHX-nanoHA disc coatings showed a slow release over time for several

days, being the amount of CHX released higher than MIC values for S. aureus ATCC 25923

(MIC=4 mg/L).

The 1500 mg/L CHX + nanoHA and 500 mg/L CHX + nanoHA coatings showed strong

anti-bacterial activity and the other coatings showed low anti-bacterial activity.

All CHX + nanoHA830 disc coatings exhibited strong anti-adhesive properties.

Different coatings were tested, showing anti-microbial activity, revealing that binding

of CHX to nanoHA discs did not affect anti-microbial activity of CHX.

These results showed that CHX + nanoHA disc coatings might be a good alternative to

medical devices implants.

Conclusions

Perform coatings surfaces characterization (XRD, AFM, zeta potential and angle

contact).

Perform more anti-microbiological tests with other relevant bacterial strains.

Perform citotoxicity tests with the coatings.

Ongoing work

Acknowledgements

Supervisor: Dr. Maria Pia Ferraz

Co-supervisor: Prof. Dr. Fernando Monteiro

Prof. Dr. Luís Melo

Dr. Olga Nunes

Dr. Cândida Manuel

All Biocomposites Group

Liliana Grenho

Ângela Carvalho

LabE007 colleagues

Project NaNOBiofilm

(PTDC/SAU-BMA/111233/2009) Funding

0,0%

29,5%

77,6%

100,0% 100,0%

0

10

20

30

40

50

60

70

80

90

100





Per

cen

tage

of

anti

-ad

hes

ive

pro

per

ty (

%)

Surfaces

Adsorption, system release and antimicrobial properties of chlorhexidine on nanohydroxyapatite...

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