Colds and surtaces 8 Soitertces 169 (2018) 72-81, Contents lists available at SlonceDirect, Colloids and Surfaces B: Biointerfaces journal homepage: www-elsevier.com/locate/colsurtb Clavanin A-bioconjugated Fes0,/Silane core-shell nanoparticles for | @®) thermal ablation of bacterial biofilms cae Kalline L. Ribeiro*, Isaac A.M. Frias”, Octavio L, Franco“, Simoni C, Dias“, Ailton A. Sousa-Junior®, Osmat N. Silva“, Andris F. Bakuzis®, Maria D.L. Oliveira*', Cesar AS. Andrade" + ronan de Pi Cradujao em novos Teraptatica, Universidade Feerl de Pemambuc, 5670-901 Ree, Baa Rede Pecan em soecolgi idwesdate Picea Det nts Nacional Ciencia Fecoli Unesdod Federale Pernombch Br “Sno Botech Ps gradu em otecreloie,Unverstate Clea Dom ose, Carpe Grate MS, Bra “stato de ca, Unwersae Feral de Gos 74500 990Cl O, Brac Departament de Bina, Unverse Federal de Perna 508 70901 Ree PE. Br ARTICLE INFO ABSTRACT ‘Accepted 25 Atl 2008 “The use of central venous catheters (CVO)is highly associated with nosocomial blood infections and its se largely requires systematic assessment of benefits and risks Bacterial contamination of these tubes fs frequent and may result in devel bial consortia also known as biofilm, The woven nature of biofilm provides 2 practical defense against antimicrobial agents. faiitating bacterial dis- semination th ‘body and development of antimicrobial resistance. In this Work. the =a auth blizng Fe;0,-aminosilan core-shell nanopar- Kepwertpetes icles functionalized with antimicrobial peptide clavanin A (lava) a8 an antimicrobial prophylactic Ghvaninh wards Staphylococcus aureus, Fscherca col, Preudamionas aeaginase and Klebsiella preumoie, Ks au antcbioflnattachmentchatactetstic relies in clavAnatural activity to disrupt the bacterial ip me= ties brane, The aninosiane shell prevents ion leaching, which isan important nuteent for bacterial growth, Hypertesma FesO,-clavasmesified CVCs showed to decrease Gram-negative bacteria attachment up 10 90% when compared to contol clean CVC, Additionally, when hypertherinal treatment i eggered far 5 min a 80°C ina tubing that already presents baceval biofilm {CVC-BF, the ability of attached bacteria reduces up {0 88%, providing an efficient solution to avold changing catheter ‘©2018 Elsevier BY. Alright reserved, 1. Introduction, Central venous catheters (CVCs) are customarily used as vascu- lar access and venous pressure monitoring in renal dialysis units, intensive care units and oncology departments: however. their use ‘ean cause complications with increased morbidity and mortality, particularly n children [1.2]. Bloodstream infections (BSI are the ‘most serious complication related to CVCS, occutting in 1-5 per de ernambc, 30570901, Ree PE, ash "Email edresves Kaine ive otuine {KL Ribeire sazcmerales@utpebr AM Fay acancoouc bs (OL Franea), sores (SC Da) Sisusajtio‘gnall com (AA Sousu Juni), ona sivaeeatoenedbe {ON Siva). sakiaseurg oc (AR Bakuas} m.canellyepecnpg>r (MDL Olvera ‘raneraeedpqenpabr iC. Andrade} 1000 catheter-days |3), and the cost per case varies in tenths of thousands USD [4), Important sterile precautions by inserters sich as skin antisepsis prior to insertion and early catheter removal are some of the most popular actions reported to effectively reduce (CVCS-BSI5) CCVCS-BSI derive from bacterial attachment to an irregular sur- face that slowly promotes biofilm formation. Before the alarming, emergence of drug-resistant bacteria, antibiotic impregnated CVS were proposed to reduce catheter colonization rates (|. Con- sequently, other alternatives had been explored as the use of antimicrobial peptides (AMPs) such a5 defensins, cathelicidin, ‘ecropins, clavanins, among others (7), A alternatives, catheters have also been treated with several anti-microbial coatings to prevent infection [8.9], and simple dip-coating or grafting of polymers have shown to increase biocompatibility besides extend- Ing catheter life time and avoiding bacterial attachment (10,Kibo a Cll ond Sac: 8 Bointerces 169 (2018) 72-81 n Furthermore, dual functional antibiofilm and antifouling copoly ‘mer coatings [11.12], antisadhesive antimicrobial coatings || temperature-sensitive switchable bactericidaljcell repellent sur faces |, and composite catheters for near-infrared imaging have been described (15). Some authors have reported methodologies for active removal of infectious biofilm. For instance, Levering etal [16 developed an active deforming catheter capable of mechani cally removing infectious biofilms. Despite the inconveniences of using iron-based nanoparticles, Geilish ef al. encapsulated iron oxide in polymersomes as nanocarriers for successful biofilm eradication by action of the catalytic decomposition of hydo- zen peroxide |17|, Besides iron oxide nanoparticles (Fe,0,, NPs), ‘other metal or metal-oxide NPs and carbon-based nanomaterials hhave also shown antibacterial properties |18,19), however they should be used thoughtfully since their effects on human health ae still largely unknown (20). Despite these concerns, Fe;04.NPS have also been successfully used in imaging and thermother~ apy applications such as cancer therapy and controlled burst of ‘rug-carriers|21-22], Specificbiomedical applications of magnetic core)biofunctional shell nanosystems are largely addressed in spe~ cialized reviews [2425]. Nanotechnological applications of mild {emperature hyperthermia focus on locally injected or systemically administered nanoparticles that are activated by exterior enei sources to generate heat (25). More recently, our group perlected the precise determination of heat delivery during in vivo magnetic hyperthermia (27), Hyperthermia corresponds to a therapeutic procedure relatedto increase in temperatures below 50°C whereas {emperature processes above 50°C are considered thermal abla- ‘ion. Of nate, AMPs are small molecular mass peptides (lower than 50 amino acid residues) that, in general, present high thermal ta- bility [28]. In this context, the combination of both, peptides (eg. AMP) and photothermal responsive iron oxide nanoparticles is of great applicability also interesting when employing peptides that hhave been engineered to polymerize at specific temperatures [25 ‘The nanotechnological novelty of this approach combines the antimicrobial properties of clavanin A (clavA), a marine tunicate-derived AMP, with photothermal responsive iron oxide nanoparticles to extend CVCS life time and, when desired, activate thermal ablation to begin with bacterial inactivation. Core/shell wrapping begins by treating the tubing with 2 plasma cleaner in air atmosphere to promote surface hydrophilicity that enhances the attachment of aminated aminopropyl timethoxysilane (APTS) ‘Afterwards the shell is functionalized by dimercaptosuccinic acid (Fes04-DMSA) which enables carboxyl groups for further biocon- juugation [20]. CVC-NHy was dip coated in the Fes04-DMSA NPs solution, Activation with EDC/NHS and simultaneous silanization Df APTS on the catheter promotes Fe,04-DMSA immobilization. At the same time, activated carboxyl groups covalently bind clavA, creating the bioactive shell. Modified CVC with Fe,0,-clavA bio- conjugated material was tested for growth inhibition on bacterial cultures of Staphylococcus aureus, Escherichia col, Pseudomonas aeruginosa and Klebsiella pneumoniae, all main opportunistic bac- {eria that lead nosocomial infections among hospitalized patients. Since a silane shell envelops FesOq core, anti-biofilm-attachment action relies directly in the natural activity of elava to disrupt the bacterial lipid bilayer, and no evidence from hydrogen peroxide action was found, ‘The involvement of AMPs in controlling bacterial infection is, well documented (31, and in fact, their use is encouraged to cur= tall the incidence of multidrug-resistant infections (32). Clavanin A has a distinctive in vitro antimicrobial activity towards Gram- positive and ~negative bacteria, and because ofthe high cholesterol content of the mammalian cell, no eytotoxic activities have been. observed against human cells or acute toxicity tests at any con- centration /23), Specifically, clavA reduces the mortality of mice infected with E coli and S. aureus in 80% [34), The surface modifi- cation of biomedical devices is well related in specialized reviews [935]. Nanomaterials based in metals such as copper, zinc, ti ‘nium, magnesium and gold have been developed in recent years In particular, the inclusion of silver nanomaterials is considered ‘one of the most promising strategies to evade bacterial infections [25.37], However, over clinical trials, slver-containing CVCs have not shown significant effec in controlling BSI |38|, For that reason, there is the need to test new materials and alternatives to improve the prophylactic or counteracting activity of CVCs towards BSL 2. Materials and methods 2.1, Materials and reagents ‘on (i) chloride hexahydrate. iron (Ut sulfate heptahydrate, (3- _aminopropyl) trethoxysilane (APTS), ammonium hydroxide, meso 23 dimercaptosuccinic acid (DMSA) dimethyl sulfoxide (DMSO), 1-ethyl-3-{3-dimethylaminopropyl) carbodiimide (EDC) and N- hydroxysuccinimide (NHS) were purchased from Sigma Aldrich (St. Louis, USA}, Double himen central venous catheters were obtained from Arrow International, Inc. (Reading, PA, USA), Clavanin A (high Putity> 95%) was synthesized by Peptides 2.0 (USA) using the solid phase following theN-S-fluorenyimethyloxycarbonyl(Fmoc)strat- egy with 95% purity degree. Mass and sequence was checked by using MALDI ToF. 22. Synthesis and chemical modification of Fe304Nps-DMSA Fe,O4Nps were prepared according to a method previously described by our group |39]. Briefly, a NHOH solution (1.5 M) was slowly added under stirring toa FeSOy:FeCl, solution (0.1 M:0.5M), ‘until 2 dark solution was obtained (pH 8) ané maintained under stirring for 12h, The resulting magnetic particles were magneti- «ally separated, For chemical modification, Fe;0,4Nps (0.01 g) were dispersed in a DMSA solution (0.3M) prepared in DMSO (10 mL) and stirred at room temperature for 24h. The mixture was washed 5 times with ethanol under magnetic separation and the particles were dried at 60°C for 12h, 23. Bioconjugation ofPesO,Nps-DMSA-clavA and surface modification of silicone catheter Catheter’s surface modification was performed according to a previous report (0), with a slight modification on the size of the segmented substrate (0.5 cm). Briefly, the tubing was submitted to an air plasma treatment for Smin in a SW PDC-32G Harrick plasma cleaner (Ithaca, NY) and then immersed in a APTS solution '1M) in anhydrous ethanol overnight to obtain primary amino ‘groups exposed on the surface. Afterwards, silanized segments were thoroughly rinsed with ethanol and blow-dried with nitrogen, {g28 al 100m temperature. Finally, the substrates were immersed in 4 glutaraldehyde solution (2.5%) for 10min and immediately trans- ferred to the FesOq-DMSA-clavA solution. Initially, FeyOq-DMSA, NPs (0.01 g) were activated in an EDC: NHS (0.4 M:0.1 M) solution for 10-min and, subsequently, washed with phosphate buffer sola- tion (PBS, pH 7.4) under magnetic separation. Then, ClavA solution mL at 0.01 mgmL-) was added and maintained for 10h under Stirring at room temperature 24. Morphological analysis The structure of FesOqNps was evaluated with a transmission electron microscopy (TEM) FEI Morgagni 268D (FEI, Eindhoven, The Netherlands) operated at SOKV. The morphology of the catheter surface was analyzed before infection, during infection and after thermal ablation with 2 scanning electron microscopy (SEM) (EOL1” AL Rb a / Calis and Sues: Boies 169 (2018) 72-81 J8MB610, Japan). ASEM samples were prepared in aSCDQSO sput- ter coater (Bal-Tec Balzers, Liechtenstein) for the deposition of a ‘Genm thick Au film, 25. Antimicrobial activity testing of Fe, Oq-clavA-modified catheter To evaluate the antimicrobial activity of modified catheter against $. aureus, K pneumoniae, E.coli and P. aeruginosa, we per ormed a modified growth inhibition assay by broth microdilution using @ microtiter plate assay according to the National Commit- tee for Clinical Laboratory Standards (NCCLS, 2003). All bacteria ‘were grown from freezer stocks in Luria-Bertani (UB) broth at 37°C overnight. The cultures, were subcultured, grown and adjusted 0 a concentration (colony-forming unit, CFU) of approximately 5% 105 CFUmL"! determined by ODsoo readings. All coated and uncoated CVC segments (N= 3) were disinfected by submerging in 70% ethanol solution for 10min, a well-known protocol that dis- infects by denaturing the bacterial membrane [41 |, and to which resistance has never been reported [42]. Then, the segments were tinged three times with sterile LB broth, and, subsequently, each segment was placed in microcentrifuge tube containing 1mL of LB broth adjusted at”5 x 10 CFUsml~! bacterial concentration. All tubes were placed at 37°C at 50 RPM on shaker for 6h. In ‘order to count the amount of bacterial growth we followed cur tent protocols in microbiology |43), After incubation, planktonic bacteria growth was assessed performing serial dilutions for drop plate methodology (44). For instance. 100 L of bacterial culture ‘were serially diluted and spot plated for. To count adhered CFUs, ‘each catheter segment was rinsed three times with sterile LB broth, and transferred into microcentrifuge tube containing sterile PBS (500 L) and set for 10min into a 100W, 42 KHz sonicating bath, Finally, each sample was vortexed at high speed for 10s, serially diluted and spot plated for quantification. 26. Thermal ablation testing of Fe,04-DMSA-clavA-contaminated catheter Coated and uncoated tubing samples were submersed in 70% ‘ethanol for 10min. Then, the segments were rinsed three times ‘with sterile LB broth. Each segment was placed in microcentrifuge tube containing 1mL of LB broth adjusted to a concentration of approximately 5x 10° CFU.mL~ of each bacterium and incubated at 37°C in a shaker at 0 RPM for Gh. For photchyperthermia (PHAB) assays, ll CVC segments were rinsed three times with ster ile PBS and exposed for S min to an 808-nm laser at different power ‘outputs to achieve different average temperatures (50, 60, 70 and °C), The photothermal experiments used a diode laser with ‘B081nm wavelength, model Laser iZi 808, bought from LASERline (Sao Paulo, Brazil) with 1000 mW output power, The temperature ‘was monitored using an infrared thermal camera bought from FLIR, ‘model $C 620 (Wilsonville, OR). Subsequently, PHAB treated seg- ments were placed in microcentrifuge tubes containing TmL of sterile LB and incubated at 37°C in a shaker at 50 RPM. The broth ‘was refreshed every 24-h for 4days by replacing half the existing ‘medium with sterile LB, Panitonic cells were counted by taking 100 LL. of daily bacterial culture which were serially dikited and spot plated for CFU counting. All plates were incubated at 37°C ‘overnight or until visible colonies formed, 27. Evaluation of antimicrobial activity of es04-DMSA-clavA-modified catheter in vive in a mouse skin infection model ‘Animals (CS7BL6 mice weighing 18-22 g) were provided by the ‘Central Bioterium ofthe Catholic University of Braslia-UCB.Allani- ‘mals were housed in individual cages under aconstant temperature (22°C) and humidity with a 12-h light/dark cycle and had access {food and water ad libitum throughout the study. The mice were euthanized by CO) inhalation at the end of the experiments. All procedures, care, and handling of the animals were approved by the Ethics Committee of the Catholic University of Brasilia number (005/13, Murine skin infection model was performed as previously described by Yu and collaborators with minor modifications [1 Mice (N=8 per group) were anaesthetized, their dorsal surface was shaved and the surgical area was disinfected with chlorhex- ‘dine digluconate 2x (Rioquimica, Brazil) An incision (05cm) was performed in the dorsal surface, and then 10 aL of K. pneumoniae suspension (1 x 10% CFUmi-!; previously cultured as described by Silva and collaborators) (34) was introduced into the incision, catheter fragments (40 mm) were inserted in the incision and the skin was closed witha silk suture, Mice were euthanized at 7 days. post-surgery, the wounded muscle tissue was excised, weighed and half of it was homogenized in 1 mL of PBS. Serial dilutions of the homogenates were plated on BHI agar in tiplicate (Himedia, India), and the results were expressed as CFUmt™ 28. Cytokine assays and statistical analysis Supernatant of homogenized wounded muscle tissue (obtained as described in the previous section) was used after bacterial counting, Homogenates were centrifuged at 10,000rpm for 10min, and supernatants were removed and held at =20°C unsil analy- sis. Gamma interferon (IPN-), interleukin-S (IL-6), I-12p70, and umor necrosis factor alpha (TNF-a) were measured using an enzyme-linked immunosorbent assay (ELISA) kit according to the ‘manufacturer's instructions (RED Systems, Minneapolis, MN, USA), Application of sterile PBS was used as negative contro! forthe anal- ysis For statistical analysts, the data is presented as mean standard eviation (SD) of values and submitted to one-way analysis of vari- ance (ANOVA) followed by Tukey's test, P-values of <0.05 were considered significant. Cytokine levels were expressed as percent age change relative to negative control. GraphPad Prism software v5.0 (GraphPad Software) was used forall statistical analyses 3. Results and discussion 3.1. Morphological studies ‘The strategy followed to functionalize the CVC segmentsisillus- trated in Fig. |. A more detailed microscopy of coated Fe,0Nps performed in TEM (supplementary material Fig. 51) shows homo- ‘Reneous cistribution of spherical nanoparticles with an average size of 10nm. High-resolution TEM permitted to appraise latice fringes congruous to a typical cubic spinel crystal structure. By sing the free software Image [45], we performed a Fast Fourier ‘gansform (FFT) to estimate the lattice spacing as 042m, a is- ance assigned to the (021) lattice plane of Fe,0, by a previous report [46 The catheter segment was initially treated with air plasma to develop a hydrophobic surface that contributes to the seli- assembly of an APTS film, an strategy followed in various processes involving alkoxysilane immobilization (Fig, 2) 47], The change in morphology of the clean CVC surface (Fiz 2a) after incubation fn the Fe304-clavA nanoparticles solution (Fis. 2b) demonstrates ‘hat the immobilization occurred successfully. Large nanoparticle densities result in higher magnetic moments (Fig. 2c): different netic moment hysteresis curves are found for samples A, B, Cand control. Note that at higher flelds one can clearly observe a diamagnetic contribution (due to the catheter material). The con- {rol signal shows contribution from the sample holder and theKibo a /Cllod ond Sac: 8: Binterfces 169 (2018) 72-81 1s eve catneter pers plasma meaiieaton staraenyse pb fo0cousncion Fig, Moieation process fz catheter sezment wih plasma and APTS followed by gutarakiehyde-2ctivated Fy O4-DMSA-) an average temperature profile a the surface of otal abe moded eather 20x10" ‘5x0? s.oxtor sox S. aureus CFUImL E.coli CFUImL 00 40x10 30x10" ' + oxo! pat .oxto? P. aeruginosa CFUImL. K. Preumoniae CFUImL. Fig. 6 Care of plankton. surar( Ect) Peerage) and K preumoniae ring 96ncukure, er hyperthermia appisions (sonKL deo era Clots and Surfaces: ines 189 (2018) 72-81 " tb Log ° CFU mL* of e invive ns mouse skin infection model indkses P= 000, and in vivo immunomodulatory activities through the reduction, 1 the release of pro-inflammatory cytokines |58], Fe04-DMSA~ clavA-modified CVC reduced the IFN-y 45%, TNFa 72% and Il-12 155% levels, while the IL-6 level was reduced in 82%, On the other hhand, te presence of CVC did not appear to interfere with the pro- duction of any of the cytokines analyzed here, It is worth noting that the levels ofcytokines detected in treated wounds are notlarge enough to cause deregulation of the immune response. However. they are present at a satisfactory level required for clearance of bacteria in cutaneous infections (60,51) ‘To the best of our knowledge, the combination of the non- antibiotic AMP clavA with factional material stich as FeO, tha, besides its antibiofiim properties avails photothermal properties. hhas not been previously attempted, These interesting results sug- est a promising approach for treating or preventing CVC-BSI in cases where the risk of exchanging catheters outweighs the tisk of controlling the substrate’s infection. However, further studies regarding the lasting of ts activity within more days or weeks, are {deal to assess it clinical functionality. ‘Our findings are similar to those of commercial nitrofurazone and silver coated catheters (52.53), however, while their activity ‘decreases with time, our approach assures photothermal ablation application when needed. In the present study, we maintained Smin of thermal treatment but better achievements might be obtained by increasing the time of treatment. Of note, high tem- peratures can also hatm the tissues surrounding the implants However, the risks are to be curtailed by employing short laser pulses applied at different output powers, thus inducing instanta- neous heat dissipation processes [64]. This procedure is known as a duty cycle strategy, where duty is the ratio between the time of Pulse ON to that of Pulse ON+ Pulse OFF, A detailed study of ther= ‘mal dose is beyond the scope of the present article and will be presented in future reports, however, itis known that decreasing, ‘the duty cycle can avotd harm to the tissue around the implant as previously reported by Decorato et al. [95]. Other strategies ‘exploit a distinct temperature range around 40° (even at duty=1), Which does not induce any damage to the surrounding issue due to heat transport. At this low temperature range, heat alone can-90 AL Rb a Caled and Suc: Boies 169 (2018) 72-81 not destroy the biofilm: however, the temperature can trigger the release of therapeutic agents to treat the biofilm. This delivery strat- ‘egy has recently been used to treat biofilms with thermal-sensitive liposomes loaded with antibiotics (55,57), and one can imagine a similar approach with our catheter by using its magnetic proper- ties to attract heat sensible nanocomposites Finally, both strategies requite the use of fiber-optic lasers. These kind of devices have become popular and are easy to find [68 for example the Visu- alase laser ablation system (Medtronic) oF the ClearPoint system (MRL interventions) [69], one should only choose an adequate laser ‘wavelength that ensures interaction of the non-ioniing radiation with the targeted nanostructures. 4. Conclusion In this paper, we presented a simple yet rounded strat- ‘egy that employs a bifunctional antimicrobial clavanin A/Fe,0, hybrid nenosystem for 808-nm-light-mediated ablation of bacte- ‘ial biofilm to improve the prevention of central venous catheter contamination and its associated risks. CVC tubing was modified bby immobilization of core/shell Fe;Os/APTS nanoparticles bio- conjugated by DMSA with AMP clavanin A. The proposed tube ‘coating had excellent antimicrobial activity, inhibiting the attach- ‘ment of Gram-negative bacteria (70-90%) and moderate activity ‘over Gram-positive bacteria (45%). In addition, the count of plank tonic bacteria was slightly diminished by up to 20%. These results are interesting since the functionalized antimicrobial shell prevents bacterial adhesion by the disrupting action of clavA, and therefore, decreasing the risk of biofilm formation and possible blood stream infections. When CVC tubing has already grew bacterial biofilm, we showed that the photothermal properties of iron oxide nanopatti doh Stpie sb saps’, number: 27138 189] ON siva.¢ ela uereeaner, EF Haney, eM. Fenstersefes,SMRibeire, Wr Porte. own C Frasier. TMB Rezede Se Moree, TX EW. Hancock OL France Anannfective spec ppt wih a) nical andimmuneredaltory artis. 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