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 metall meta 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 Cazzola 1 , Sara Ferraris 1 , Valeria Allizond 2 , Giuliana Banche 2 , Cinzia Margherita Bertea 2 , Giovanna Gautier di Confiengo 3 , Chiara Novara 1 , Andrea Cochis 4 , Lia Rimondini 4 , Silvia Spriano 1 martin ina.caz a.cazzol ola@ a@pol olit ito. o.it it 1

ISSUE: bacterial infections in biomedical devices Antibiotics Biofilm Bacterial administration/ Infections formation contamination removal of the implants  Epidemiological impact (morbidity and mortality)  Economic impact (prolonged time of hospitalization, loss of working days, increased use of diagnostic procedures, removal of the implant) 2

SOLUTIONS? - Development of Antib ibio ioti tic The e systemic mic resistant bacterial strains ther erapy use e leads ds to: - Side effects Antim imibr ibrobia obial Com ompos positi tion on Mode de of actions ions coati tings gs Anti-adhesive or Surface modifications bacterium-repellent (chemistry/Tophografic) Inorganic surfaces Immobilization of Synthetic Bactericidal / anti- antibacterial agent Organic adhesive surfaces Incorporation and Es Essential ential oils Natural Bactericidal systems release of based on release antimicrobial agent

ESSENTIAL OILS 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) COMPOSI OSITION TION ➢ TERPENS (monoterpens e sesquiterpens) ➢ TERPENOIDS (alcohols, aldehydes, chetons, esters, oxides) ➢ PHENYLPROPANOIDS (coumarins, fenilpropeni, hydroxycinnamic acids) ➢ SULPHUSES AND NITROGEN COMPOUNDS 4

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 5 aureus ,” Evidence-Based Complement. Altern. Med., 2015, vol. 2015, pp. 1 – 9.

Aim of the work ➢ 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 of 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 onali 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. 6

MATERIALS AND METHODS SAMPLES: ➢ Ti6Al4V ALLOY ➢ Ti6Al4V ALLOY CHEMICALLY-TREATED (CT) ➢ Stainless steel 316L SYNTHESIS METHODS PROCEDURES FOR THE ANALYSIS Washing hing ➢ H 2 SO 4 ➢ NaOH ➢ degreaser Funzional ionalizz izzaz azione one Release se in wat water er 3 h ➢ ➢ 7 h Water 3 days ➢ ➢ 7 days 7

CHARATCERIZATIONS OF THE COATINGS ➢ Fluorescence microscopy (mint autofluorescence) Coating ing ➢ XPS ➢ FTIR ➢ Tape test (coating adhesion) ➢ Antibacterial test 7mLwater with E.col oli (10 3 cells/mL ) Coating ing after wa water er wa washi hing ng ➢ FTIR ➢ Tape test Functional nctionaliz ization ation alamar blue Cfu count XPS 8

Fluorescence microscope observations Ti Ti_mint Ti _mint_rel3h 100µm 100µm 100µm Ti _mint_rel7h Ti _mint_rel7d Ti _mint_rel3d 100µm 100µm 100µm 9

Fluorescence microscope observations steel steel _mint steel _mint_rel3h 100µm 100µm 100µm steel _mint_rel3d steel_mint_ril7h steel _mint_ril7d 100µm 100µm 100µm A fluorescent layer is visible on both Ti alloy and steintess steel samples coated with mint oil, 10 also after 7 days of release in water

FTIR mesurements on steel samples Peaks ks Assigmeme igmement nts 3450 3450 – 3200 00 Stretching C-H stretchi retching ng – OH OH C=O Bending C-H (aliph), cm −1 cm (aliph) stretching C=C (arom) 2960 2960 – 2870 70 stretchi etching ng C – H cm cm −1 * 1780 – 170 700 stretchi etching ng C=O C=O cm −1 cm * steel_mint ance [A.U] bendin ding g C – H (H 2 SO 4 ) steel_mint(H2SO4) 1450 – 13 1370 (aliph ph), ), ectanc * steel_mint(NaOH) reflect cm cm −1 stretchi etching ng C=C steel_mint(deg) (arom) * 3800 3400 3000 2600 2200 1800 1400 1000 600 Wavenum number [cm -1 ] The spectra of all the samples are characterized by the typical vibrational peaks of oxygenatedmonoterpenes 11

Tap ape e Test st (A (ASTM TM D 33 3359) 59) a. Cross ss-cut cut area b. T Tape prepa parati ration posi siti tioni ning ng c. Tape d. Observati tion removal 12

Tap ape e Test st (A (ASTM TM D 33 3359) 59) Before tape test After tape test Mint coating on polished steel: Classification 2B 100µm 100µm Before tape test After tape test Mint coating on high roughness steel: Classification 3B 100µm 13 100µm

XPS on functionalized samples CT CT_50%mint(funz) (atomic omic%) %) 31.25 26.12 C-C, C-C, 2.00E+04 2.00E+04 on HR carbon C-H C-H 1.50E+04 1.50E+04 C/s C=O C=O 1.00E+04 C=C 1.00E+04 C=C C-O C-O 4.00E+03 4.00E+03 298 296 294 292 290 288 286 284 282 280 298 296 294 292 290 288 286 284 282 280 Binding Energy (eV) Binding Energy (eV) (atomic omic%) %) 49.16 45.6 .64 1.20E+05 1.40E+05 1.10E+05 Ti-O 1.20E+05 Ti-O 1.00E+05 gen HR oxygen 9.00E+04 1.00E+05 8.00E+04 C/s C/s 8.00E+04 7.00E+04 Ti-OH 6.00E+04 6.00E+04 HR Ti-OH 5.00E+04 4.00E+04 4.00E+04 C-O, 3.00E+04 C=O 2.00E+04 2.00E+04 1.00E+04 0.00E+00 544 542 540 538 536 534 532 530 528 526 544 542 540 538 536 534 532 530 528 526 14 Binding Energy (eV) Binding Energy (eV)

Antib ibact acteria erial test E.coli viability 24h E.coli viability 48h 2500 2500 * * 2000 2000 O.D. (@ 490 nm) O.D. (@ 490 nm) 1500 1500 1000 1000 500 500 0 0 steel steel_mint steel steel_mint E.coli viability (vs time) The mint coating was able to reduce the 2500 amount of adherent metabolically active bacteria in a significant manner in O.D. (@ 490 nm) 2000 comparison with bare control materials steel * * after 24 and 48 h of incubation 1500 steel_mint 1000 24 48 Hours 15

CONCLUSIONS  The su succes cess s of fun uncti tion onaliz 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 of 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 of 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 on 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. 16