metall meta llic ic bio iomat materia erials ls wi with es - - PowerPoint PPT Presentation

Su Surfac ace e coat ating ing an and funct nctionaliz ionalizat ation ion of meta metall llic ic bio iomat materia erials ls wi with es esse senti ntial al oil ils s for r an antibact ibacteria erial l ap appli plications cations

Martina Cazzola1, Sara Ferraris1, Valeria Allizond2 , Giuliana Banche2, Cinzia Margherita Bertea2, Giovanna Gautier di Confiengo3, Chiara Novara1, Andrea Cochis4, Lia Rimondini4, Silvia Spriano1

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ISSUE: bacterial infections in biomedical devices

 Epidemiological impact (morbidity and mortality)  Economic impact (prolonged time of hospitalization,

loss of working days, increased use of diagnostic procedures, removal of the implant)

Bacterial contamination Biofilm formation Infections Antibiotics administration/ removal of the implants

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SOLUTIONS?

Antib ibio ioti tic ther erapy The e systemic mic use e leads ds to:

• Development of

resistant bacterial strains

• Side effects

Incorporation and release of antimicrobial agent

Antim imibr ibrobia

• bial

coati tings gs

Surface modifications (chemistry/Tophografic) Immobilization of antibacterial agent Com

• mpos

positi tion

• n

Anti-adhesive or bacterium-repellent surfaces Bactericidal / anti- adhesive surfaces Bactericidal systems based on release Es Essential ential oils Inorganic Organic Natural Synthetic Mode de of actions ions

ESSENTIAL OILS

COMPOSI OSITION TION ➢TERPENS (monoterpens e sesquiterpens) ➢TERPENOIDS (alcohols, aldehydes, chetons, esters, oxides) ➢PHENYLPROPANOIDS (coumarins, fenilpropeni, hydroxycinnamic acids) ➢ SULPHUSES AND NITROGEN COMPOUNDS DEFI FINITION NITION: product obtained from

a natural raw material of plant origin, by steam distillation, by mechanical processes from the epicarp of citrus fruits, or by dry distillation, after separation of the aqueous phase by physical processes(ISO/D1S9235.2)

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MECHANISMS OF ACTION OF ESSENTIAL OILS

• J. C. Lopez-Romero et al., “Antibacterial Effects and Mode of Action of Selected

Essential Oils Components against Escherichia coli and Staphylococcus aureus,” Evidence-Based Complement. Altern. Med., 2015, vol. 2015, pp. 1–9.

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➢The relevance of the topic of this work deals with the need for innovation in the de devel elopm pment ent of anti-adh dhesi esive e surface aces, since a range

• f medical, laboratory and general purpose instrumentation requires

antibacterial safety. ➢To this aim, stainless steel 316L 6L and T d Ti6 i6Al4 l4V, which are alloys used in the fabrications of different devices, were coated ed or functi ctiona

• nali

lize zed d wit ith Menth tha pip iperit ita es essen entia ial l oil ils. ➢The procedure of coating was developed for non-implantable device, while functionalization of the surface of chemically treated Ti6Al4V (CT) was developed for implant applications in contact with bone.

Aim of the work

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MATERIALS AND METHODS

SAMPLES: ➢Ti6Al4V ALLOY ➢Ti6Al4V ALLOY CHEMICALLY-TREATED (CT) ➢Stainless steel 316L

SYNTHESIS METHODS

Funzional ionalizz izzaz azione

• ne

Washing hing

Water

Release se in wat water er

➢H2SO4 ➢NaOH ➢degreaser ➢ 3 h ➢ 7 h ➢ 3 days ➢ 7 days

PROCEDURES FOR THE ANALYSIS

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CHARATCERIZATIONS OF THE COATINGS

XPS ➢ FTIR ➢ Tape test Coating ing Coating ing after wa water er wa washi hing ng Functional nctionaliz ization ation alamar blue Cfu count

7mLwater with E.col

• li

(103 cells/mL)

➢ Fluorescence microscopy (mint autofluorescence) ➢ XPS ➢ FTIR ➢ Tape test (coating adhesion) ➢ Antibacterial test

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Fluorescence microscope observations

100µm 100µm 100µm 100µm 100µm 100µm

Ti _mint_rel3d Ti _mint_rel7d Ti _mint_rel7h Ti _mint_rel3h Ti_mint Ti

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Fluorescence microscope observations

steel _mint

100µm 100µm 100µm 100µm 100µm 100µm

steel steel _mint_rel3h steel_mint_ril7h steel _mint_ril7d steel _mint_rel3d

A fluorescent layer is visible on both Ti alloy and steintess steel samples coated with mint oil, also after 7 days of release in water

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FTIR mesurements on steel samples

Peaks ks Assigmeme igmement nts 3450 3450–3200 00 cm cm−1 stretchi retching ng –OH OH 2960 2960–2870 70 cm cm−1 stretchi etching ng C–H 1780–170 700 cm cm−1 stretchi etching ng C=O C=O 1450–13 1370 cm cm−1 bendin ding g C–H (aliph ph), ), stretchi etching ng C=C (arom)

The spectra of all the samples are characterized by the typical vibrational peaks of

• xygenatedmonoterpenes

600 1000 1400 1800 2200 2600 3000 3400 3800

reflect ectanc ance [A.U] Wavenum number [cm-1]

steel_mint steel_mint(H2SO4) steel_mint(NaOH) steel_mint(deg)

Stretching C-H (aliph) C=O Bending C-H (aliph), stretching C=C (arom)

* * * *

(H2SO4 )

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Tap ape e Test st (A (ASTM TM D 33 3359) 59)

• b. T

Tape posi siti tioni ning ng

• c. Tape

removal

• d. Observati

tion

• a. Cross

ss-cut cut area prepa parati ration

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Tap ape e Test st (A (ASTM TM D 33 3359) 59)

Mint coating on polished steel: Classification 2B Before tape test

100µm

After tape test

100µm

Mint coating on high roughness steel: Classification 3B Before tape test

100µm

After tape test

100µm

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XPS on functionalized samples

4.00E+03 1.00E+04 1.50E+04 2.00E+04 280 282 284 286 288 290 292 294 296 298

C/s Binding Energy (eV) Binding Energy (eV)

280 282 284 286 288 290 292 294 296 298 4.00E+03 1.00E+04 1.50E+04 2.00E+04

1.00E+04 2.00E+04 3.00E+04 4.00E+04 5.00E+04 6.00E+04 7.00E+04 8.00E+04 9.00E+04 1.00E+05 1.10E+05 1.20E+05 526 528 530 532 534 536 538 540 542 544

C/s

Binding Energy (eV)

0.00E+00 2.00E+04 4.00E+04 6.00E+04 8.00E+04 1.00E+05 1.20E+05 1.40E+05 526 528 530 532 534 536 538 540 542 544

C/s Binding Energy (eV)

HR carbon

• n

HR HR oxygen gen CT CT_50%mint(funz)

Ti-O Ti-OH Ti-OH Ti-O C-O, C=O C=C C-C, C-H C-C, C-H C=O C=O C-O C-O C=C (atomic

• mic%)

%) 26.12 31.25 (atomic

• mic%)

%) 49.16 45.6 .64 14

Antib ibact acteria erial test

500 1000 1500 2000 2500 steel steel_mint

O.D. (@ 490 nm)

E.coli viability 24h

500 1000 1500 2000 2500 steel steel_mint

O.D. (@ 490 nm)

E.coli viability 48h

1000 1500 2000 2500 24 48

O.D. (@ 490 nm) Hours

E.coli viability (vs time)

steel steel_mint

* * * *

The mint coating was able to reduce the amount of adherent metabolically active bacteria in a significant manner in comparison with bare control materials after 24 and 48 h of incubation

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CONCLUSIONS

 The su

succes cess s of fun uncti tion

• naliz

lizat ation ion and coati ting ng treatments was highlighted by XPS or FTIR analysis that showed the presence of different biomolecules of the mint oil on the surface

• f the samples, according to the different procedures.

 The coati

ting ng on both stainless steel and Ti6Al4V samples resulted stable le also after 7 days

• f soaking in water, as observed by fluorescence microscopy. Moreover, the tape test

performed on steel samples showed a good stability of the coating which was increased by a higher roughness of the surfaces before the coating procedure.

 The coated steel samples were also washed with acid and basic solutions in order to test

the resistance to cleaning and analyzed by means of FTIR spectroscopy which showed that the coati ting ng resis ists ts to basic ic cleane ner r and has only a little damage after acid cleaning.

 The mint coating on steel showed the ability to reduc

duce e the e E. coli sur urfa face ce contamina ntaminati tion

• n

highlighting an anti-adhesive behavior.

 The procedure of coating and functionalization performed with an essential oil seems to

be a promis mising ing strategy egy to exploi loit t their eir antiba ibact cterial erial activi vity ty for both non-implantable and implantable applications in biomedical field and it can be easily extended to other essential oils.

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