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Differential Adhesive & Bioactive Properties of Polymeric Surface Coated with Graphene Oxide Thin Film Sudhin Thampi, A Maya Nandkumar, Vignesh Muthuvijayan, and Ramesh Parameswaran ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14863 • Publication Date (Web): 16 Jan 2017 Downloaded from http://pubs.acs.org on January 17, 2017

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ACS Applied Materials & Interfaces

Differential Adhesive & Bioactive Properties of Polymeric Surface Coated with Graphene Oxide Thin Film Sudhin Thampi, 1,2 A Maya Nandkumar,3 Vignesh Muthuvijayan,1 Ramesh Parameswaran2* 1 Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai 600036, India 2 Division of Polymeric Medical Devices, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram 695012, India 3 Division of Microbial Technology, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram 695012, India *Corresponding Author E - mail: [email protected]

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Abstract Surface engineering of implantable devices involving polymeric biomaterials has become an essential aspect for medical implants. Surface enhancement technique can provide an array of unique surface properties that improves its biocompatibility and functionality as an implant. Polyurethane based implants that have found extensively acclaimed usage as an implant in biomedical applications, especially in the area of cardiovascular devices still lacks any mechanism to ward off bacterial or platelet adhesion. To bring out such a defense mechanism we are proposing a surface modification technique. Graphene Oxide (GO) in very thin film form was wrapped onto the electrospun fibroporous polycarbonate urethane (PCU) membrane (GOPCU) by a simple method of electrospraying. In the present study, we have developed a simple single step method for coating a polymeric substrate with a thin GO film and evaluated the novel anti-adhesive activity of these films. SEM micrographs after coating showed the presence of very thin GO films over PCU membrane. On GOPCU surface, contact angle got shifted by ~30°, making the hydrophobic PCU surface slightly hydrophilic; while Raman spectral characterization and mapping showed the presence and distribution of GO over 75% of membrane. A reduced platelet adhesion on GOPCU surface was observed, meanwhile bacterial adhesion also got reduced by 85% for Staphylococcus aureus (Gram positive, cocci) and 64% for Pseudomonas aeruginosa (Gram negative, bacilli). Cell adhesion study conducted using mammalian fibroblast cells projected its proliferation percentage in a MTT assay, with 82% cell survival on PCU and 86% on GOPCU after 24h culture, while a study for extended period of 72 h showed 87% of survival on PCU and 88% on GOPCU. This plethora of functionalities by a simple modification technique makes thin GO films a self-sufficient surface engineering material for future biomedical applications. Keywords: Surface modification, implant, biomaterial, Polyurethane, graphene, electrospraying, electrospinning, adhesion


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1.

INTRODUCTION Functional restoration of human body is nowadays accomplished by implantation of

simple or sophisticated medical devices that involve use of biomaterials over long term. Any material to be used as a biomaterial needs to be biocompatible, and this is an issue of serious concern in the case of medical implants. Surface modification is a popular means of enhancing implant biocompatibility and host-implant interaction.1 Initiation of many interactions with the biological entities occurs at the implant surface. When untreated materials come in contact with blood or physiological fluid, untoward host responses may arise, such as thrombus formation, immune-reactions and secondary infections that could be fatal. In face of such a complicated scenario implant removal may be the only alternative to avoid further complications. So interface demands suitable modifications to ensure biocompatibility with surface treatment or coatings before implantation. Thrombotic and thromboembolic complications remain matter of serious concern with cardiovascular devices2. Thus to improve blood compatibility the strategies adopted are either material surface modification or material development itself. To improve the blood compatibility of cardiovascular devices, surface functionalization with antithrombotic agents or immobilization of molecules like polyethylene oxide (PEO), heparin, albumin, chitosan etc. have been practiced for long.3 But, due to complicated mechanism of blood material interactions, these have not been efficient in solving the problem; so need for exploration for new materials/ surface modification is very relevant and necessary. On the other hand medical devices are becoming the leading cause of nosocomial infections. Biomaterials attract lots of bacteria which colonize them and infect the surrounding tissues leading to severe device failure4. Infection may spread to other body parts bringing in further complications and clinical challenges. Adherence of bacteria either from pre-operative or post-operative environment to a device surface is the precursory step leading towards bacterial infection, 5 and formation of biofilm6. Biofilm provides needed protection to bacteria and allows it to thrive even under adverse conditions of antibacterial treatments or systemically administered antibiotics. Solution to this problem lies in the development of coatings and surface engineered materials that dissuade bacteria from attaching to these surfaces. Biocides can be incorporated into the materials or coated or covalently bonded, resulting either in its release or in contact 3 ACS Paragon Plus Environment


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killing without release of the biocide. The use of biocides in medical devices is debated because of the risk of bacterial resistance and potential toxicity. Anti-adhesive chemical surface modifications mostly target the hydrophobic feature of the materials. Topographical modifications are focused on roughness and nanostructures, whose size and spatial organization are controlled. The most effective physical parameters to reduce bacterial adhesion yet remain to be determined and could depend on a combination of factors.7 Future of medical implant surface engineering lies in providing a material with a wide variety of properties that eases the implantation process and shows compliance with the body milieu. It can be simply classified into two aspects wherein it has to ward off adhesion of foreign or biologically formed bodies and on the other hand, it needs to be cell and tissue amiable. Surfaces with super-hydrophobicity can solve first aspect i.e. prevent adhesion but most implants like dental implants, neural, orthopedic and vascular grafts needs to be integrated into the tissue to promote tissue regeneration while dissuading the adhesion of bio-entities like bacteria or platelets. Coating material and hydrophilicity has an important role to play to bring out such an aspect in surface modifications. Another added advantage of having a slightly hydrophilic surface, may reduce the injury caused during implant insertion into the patient which later causes immune response related complications8. GO with its plethora of properties may be a potential nanomaterial to be explored for surface modifying application. But owing to its method of preparation biocompatibility9 of GO has been an issue of concern in several studies for biomedical applications. Antibacterial properties of GO has been a topic of research for a long time stating both positive10 and negative opinions11, 12. Studies have been done in its freely suspended form and surface bound form, but very few studies has been reported on its differential properties as thin films bound over a polymeric surface with a focus on bacteria, platelets and mammalian cells. Apart from that to the best of our knowledge there is no reported technique to transfer GO as thin layers in a single step over polymeric substrates in absence of any additional polymer. Our strategy was to develop a surface engineering technique to modify a polymer surface by coating very thin GO nanosheets in a single step. Upon modifying the surface, we have studied its physical properties and interactions with several bio-entities like bacteria, platelets and mammalian fibroblast cells. With this intent, polycarbonate urethane (PCU) a widely 4 ACS Paragon Plus Environment

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acclaimed biostable form of polyurethane used in numerous medical implant applications was selected as a model. It has a highly hydrophobic surface which causes thrombus formation13 and bacterial adhesion14, hence was a suitable biomaterial for our study. Herein we’ve developed a simple surface engineering technique based on GO thin films for modifying this surface, henceforth reporting its characterisation and elucidating its novel functionalities. PCU was electrospun into a membrane and a modified electrospraying technique was used to transfer very thin layers of GO over its surface, without involving any harsh chemical reactions or radiation treatments. Studies carried out may be valuable in investigating physico-chemical and biointerface properties while may also pave way towards use of GO as a biocompatible material for surface engineering. 2.

EXPERIMENTAL SECTION

2.1.

Materials

Carbothane manufactured by Lubrizol Corp. as PCU, was used in this study. 12% (w/v %) of PCU was dissolved in mixture of di-methyl formamide (DMF) and tetrahydrofuran (THF) (50/50 v/v) for electrospinning. Graphite flakes (G), Minimum Essential Medium (MEM) and Hoechst 33258 were procured from Sigma-Aldrich, USA. DMF, THF, H2SO4, H3PO4, KMnO4, HCl and 30% H2O2 were purchased from Merck, India. Staphylococcus aureus (ATCC25923), Pseudomonas aeruginosa (ATCC27853), L929 cell line from American Type Culture Collection (ATCC, Rockville). Tryptic soya broth (TSB), Tryptic soya agar (TSA) and Mueller Hinton Media (MHA) was acquired from Hi-Media, India. Fetal Bovine Serum (FBS), Trypsin, Actin green 488 ready probe reagent, Penicillin and streptomycin were acquired from, Gibco, Invitrogen, USA. De-ionized water (DI/W) was used throughout this study. 2.2.

Methods

2.2.1. Electrospinning A syringe with PCU solution was loaded onto the electrospinning unit. The syringe was capped with a 21-gauge blunt-end needle (spinneret), and a positive potential was applied at the needle end with a power supply (Gamma high voltage), whereas the negative potential was connected to the target. A rotating mandrel at 2500 rpm, kept at a distance of 16 cm was used as 5 ACS Paragon Plus Environment


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collector to fabricate polycarbonate urethane membrane (PCU). The PCU solution was delivered to the charged spinneret by the syringe pump at a flow rate of 1 mL/h and electrospun at 8–10 kV potential. The process was carried out at ambient temperature (28±2°C). 2.2.2. Synthesis and Electrospraying of GO GO was prepared according to the procedure reported elsewhere.15 1g (1Eqv) Graphite flake was taken with 6g (6 equivalent) KMnO4, mixed well followed by addition of 150ml of 9:1 solution of H2SO4 and H3PO4. Stirring was done for 18h at 50°C and then cooled to room temperature. An ice solution of with 5ml H2O2 was made and the above mixture was added slowly while stirring. A mixture of shining yellow colour solution obtained was centrifuged at 6000rpm for 30 min. Supernatant was discarded and washed thrice with DI water. Washing was carried out in similar manner using 30% HCl, absolute ethanol and di-ethyl ether. The residue was dried at 50°C in a vacuum oven. The solution to be sprayed was prepared by exfoliating and dispersing GO, in DMF by tip sonication it for 45 min. Electrospraying of GO was done onto the PCU membrane by a novel solvent based method without mixing any additional polymer at 0.5kV/cm at rate of 1ml/h. The electrosprayed membranes were retrieved from the mandrel and air-dried to remove any residual solvent. 2.2.3. XPS analysis X-ray photoelectron spectroscopy (XPS) data were acquired using an Axis Ultra instrument from Kratos Analytical in the range of 1-900 eV to investigate the surface chemistries of the obtained materials. 2.2.4. SEM /ESEM Scanning electron microscopy (SEM) (Hitachi model S-2400) was used to study the morphology and topography of the membrane surface and to analyze the surface area of dispersed GO nanosheets. For analyzing surface area of dispersed GO the solution drop was placed over glass cover slip, followed by spreading and drying.

Environmental scanning

electron microscopy (ESEM) (FIE, Quanta 200) was used for observing bacterial and platelet adhered membranes. Sample preparation was done by the sputtering of thin, flat sections of the


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materials with gold. Further imageJ software was used to analyze the SEM images of GO coated glass cover slips and to calculate the average surface area of these nanosheets. 2.2.5. Raman spectra and Mapping For Raman microscopic mapping, experiments were performed with the confocal Raman microscope (Witec Inc. alpha300R). Measurements were carried out with a frequency-doubled NdYAG laser: l = 532 nm. The excitation light was polarized horizontally (in the x-direction) with respect to the Raman image. All measurements were performed using a SP2300i spectrometer and back illuminated CCD camera DU401A. The sample was investigated using a 20 x Nikon (NA=1.0) objective. The distribution of chemical species on the sample was obtained in Raman spectral mapping mode. In this mapping mode a complete Raman spectrum was recorded (integration time of 0.025s) at every mapping point (100 x 100 points for 50 x 50 µm2), leading to a 2D array of Raman spectra. Background subtraction, cosmic ray removal, averaging a certain region manually, and spectral de-mixing were performed on this 2D array of Raman spectra. Two distinct spectra for PCU and GOPCU were extracted using Cluster Analysis utilizing Witec Project plus software. In the cluster analysis method Raman images were sorted according to their similarities. As a result we get a certain number of areas or masks, which indicates where the spectra belonging to the various clusters were acquired from as well as the average spectra of each cluster. Image J an open source image processing and analysis tool was further used to analyze the GO distribution, electrosprayed over PCU membrane. 2.2.6. XRD (X-ray diffraction) XRD data were collected with a Bruker D8 Advance diffractometer with Cu K-α radiation at a scan speed of 4/min over 5–50. The GO sample was analyzed in powder form and electrospun PCU and PCU/GO composite membranes from 20 mm diameter discs. 2.2.7. Contact angle Contact angle were determined by sessile drop method using a video-assisted contact angle measuring device (DataPhysics OCA15 plus, Germany) and mapping software (SCA20 software, Germany). For getting contact angle of bulk GO, it was pelletized into 10mm discs using 4ton pressure with holding and releasing time of 1min, while for polymeric membranes rectangular 7 ACS Paragon Plus Environment


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pieces were cut. Within 10 seconds of the introduction of the DI/W droplets, the contact angle formed between the sessile droplets and the surfaces were measured. The contact angle is expressed as an average of five independent measurements taken at different GO discs and sites on each membrane. 2.2.8. Confocal profilometry Confocal profilometry has been used to study the surface profile and estimate the change in roughness before and after embedding GO. The surface roughness of PCU and GOPCU fibrous mats were evaluated with a noncontact scanning topography instrument—Talysurf CLI 1000 (Taylor Hobson) equipped with Talymap Gold 4.1 analysis software. Sample was fixed on a flat surface and 40 line profiles were obtained overlapping the edge and the surface, using a chromatic length aberration gauge. Three-dimensional images were constructed; average (Ra) and root mean square roughness (Rq) values were calculated. 2.2.9. Antibacterial property analysis Gram negative Pseudomonas aeruginosa (PA) ATCC 27853 and Gram positive Staphylococcus aureus (SA) ATCC 25923 bacterial strains were used for analysis. Agar disc diffusion method was used to check for any antibacterial leaching/diffusing property from the coated (GOPCU) and uncoated (PCU) samples. For this 6mm circular discs were cut and sterilized by immersion in 70% ethanol for 1h, followed by air drying and sterile PBS wash. Disc diffusion study was done by placing these discs over bacterial lawn on MHA medium. Gentamicin (10µg/disc) antibiotic disc was used as positive control and culture plates were incubated overnight at 35.5 ±2.5°C. Zone of inhibition (ZOI) to growth was measured to confirm any leaching/diffusing antibacterial property from the material. 2.2.10. Dynamic bacterial adhesion study 1cm×1cm PCU and GOPCU membranes were cut and sterilized by immersion in 70% ethanol for 1h, followed by air drying and sterile PBS wash. SA and PA were inoculated in TSB and allowed to grow at 35.5±2.5°C and 100rpm in a shaker incubator. Culture was harvested at log phase and brought to 108CFU/ml using McFarland’s standard 1. Dilution was made to get final bacterial count of 105 CFU/ml. Membranes, each of control and test in triplicates were placed 8 ACS Paragon Plus Environment

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into 20ml TSB with 105 CFU/ml of bacteria. These were incubated for 20-24h in shaking incubator at 35.5±2.5°C and 100 rpm. Each membrane was taken and washed thrice with PBS to remove loosely adhered bacteria then placed into sterile tube with 5 ml PBS, sonicated for 1 min followed by 1 min vortexing; this was repeated thrice to extract bacteria adhered to the membrane. The bacteria thus collected were diluted and inoculated onto Tryptic soya agar (TSA) plates in triplicates. Plates were incubated overnight at 35.5±2.5°C allowing bacteria to grow. Colonies were counted and extrapolated to CFU/cm2 of membrane. Experiments were repeated thrice, an average of at the least 9 plates was taken for each control and test membranes respectively for concluding CFU/cm2 of membrane. The membranes were transferred into new wells and fixed for overnight in 2.5% glutaraldehyde in PBS followed by dehydration by a series of graded alcohol solutions (30, 50, 70, 90 and 100%) and air dried. After that, the membranes were coated with gold for ESEM examination. 2.2.11. In- vitro hemocompatibility evaluation The hemocompatibility of PCU and GOPCU membranes were evaluated using in vitro hemolysis assay and platelet adhesion study as per ISO 10993-4. These tests were done using human blood samples in accord to the ethical guidelines approved by the Institutional Ethics Committee of Sree Chitra Tirunal

Institute for Medical

Sciences and

Technology(approval

no.:

SCT/IEC/594/2014 dated 21/04/2014). The hemolytic character of the material was assessed by means of an in vitro hemolysis test. Total hemoglobin (Hb) was analyzed with a hematology analyzer (Sysmex-K4500). Disc shape test material of 6 mm diameter in triplicate was conditioned in PBS for 5 min before exposure to blood. The samples were kept in a polystyrene plate; 2 mL of blood was added to each well, and a well without any sample was treated as a reference (Ref). A volume of 1 mL of blood was taken immediately for initial analysis and the remaining 1 mL of blood was incubated with a sample for 30 min under agitation at 70±5 rpm at 35 ± 2° C. The blood samples were centrifuged at 4000rpm for 15 min and platelet poor plasma was aspirated to prepare platelet poor plasma. The free hemoglobin (fHb) liberated into the plasma after exposure to samples was measured using UV spectrophotometer as per the equation (Eqn. 1).16 9 ACS Paragon Plus Environment


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= 1.65 × − 0.93 × − 0.73 ×

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(Equation 1)

wherein A415, A380 and A450 are absorptions at 415, 380 and 450nm wavelengths respectively. Percentage hemolysis was calculated using the formula (Free Hb/Total Hb) × 100. Hemolysis was thus calculated and results were expressed as mean ± standard deviation (SD) (n=3). A study for checking platelet adhesion was done by exposing the control and coated material to whole blood for 30min. Study was concluded by analyzing reduction in platelet count before and after exposure to material. A part of blood sample was centrifuged at 2500rpm for 5 min and platelet rich plasma (PRP) was aspirated to prepare for the platelet adhesion studies. The test materials were placed in the wells of polystyrene culture dishes and agitated with phosphate buffered saline for 5min before they were exposed to blood and PRP respectively. To the respective wells 1.5ml blood and PRP were added and 0.5ml was taken immediately for initial analysis and remaining 1ml was exposed to the materials for 30min under agitation at 75±5 rpm using an Environ shaker thermo at 35±2°C. Three empty wells of polystyrene culture dish were exposed with blood and PRP separately as reference (Ref). Blood and PRP were collected from respective initial and 30 min exposed reference and test sample wells. Platelet count was done using Haematology Analyzer (SysmexK 4500). Percentage change was calculated from thus acquired readings as per the equation (Eqn. 2).17 !"%$ =

%&%'%() *+,&'-.%&() *+,&' (.'/0 *+&'(*' %&%'%() *+,&'

× 100

(Equation 2)

After rinsing with PBS three times to remove any non - adhered platelets, the membranes were transferred into new wells and fixed for 30 min in 2.5% glutaraldehyde in PBS, dehydrated by a series of graded alcohol solutions (30,50,70, 90 and 100%) and air dried. After that, the membranes were coated with gold for examination in ESEM. All results were the average of three parallel experiments. 2.2.12. Mammalian cell adhesion and proliferation study Cell adhesion and viability of cells on PCU and GOPCU fibrous membrane were analyzed using L-929 mammalian fibroblast cells. Samples having 1×1 cm size were preconditioned for 15 min with culture medium MEM comprising 10% FBS, 100IU/ml penicillin and 100µg/ml 10 ACS Paragon Plus Environment

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streptomycin and then transferred to a fresh cell culture dish. L-929 cells were transferred to preconditioned membranes at a concentration of 1×104 cells/cm2 and incubated inside CO2 incubator for 20 min to allow cells to adhere. Additional 1 ml of culture medium was supplemented and cell seeded membranes were further incubated for 48h. After 48h in culture, the cell seeded samples were fixed using 4% paraformaldehyde, incubated overnight and washed thrice with 0.1 M PBS. The fixed samples were permeabilised by treating with 0.1% TritonX100 for 1min. The samples were rinsed with PBS and incubated with Actin green reagent for 30 min. Cell nuclei was counter-stained with Hoechst 33258. The cell adhesion was observed under fluorescence microscope exit/emis filter (A/I3) (Leica DMI 6000B). Cell proliferation was tested using the mitochondrial metabolic (MTT) activity assay. L929 cells were sub-cultured using 0.25% trypsin –EDTA and seeded to test materials at concentration of 1x104 cells/ materials and control glass cover slip, incubated for 24h and 72h. Samples at different intervals were subjected to proliferation by MTT Assay. 200ul of MTT (1mg/ml in serum free media) was added to the materials and incubated for 2h. After incubation the formazan crystals were solubilised with 400ul of isopropanol and the solution was transferred to 12well plate and absorbance was read at 570nm. Cells cultured in glass cover slip were taken as the assay control. Data presented is an average of samples in triplicates along with their respective standard deviations (SD). 2.2.13. Statistical analysis Statistical analysis were performed with Microsoft office using the excel module. Differences among the PCU and GOPCU membranes were assessed using analysis of variance (ANOVA) combined with Tukey analysis. Data values are presented as mean ± standard deviation (SD) and p-values of 0.05 and below were reported as being significant. 3.

RESULTS AND DISCUSSION

3.1.

Surface analysis using XPS

The peak areas calculation of C and O elements from XPS spectra analysis in fig.1(a) revealed that the ratio of C and O atomic element decreased from 9.84 in G to 0.63 in GO. These results confirmed the oxidation of G. XPS spectroscopy of G showed peak at 284eV attributed to C=C 11 ACS Paragon Plus Environment


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versus the oxidized samples which showed peaks at 284.4 and 286.8 eV in the case of GO, which can be attributed to C–C, –C–OH functional groups, respectively.18 The C1s spectra of G and GO are presented in fig. 1(b) and (c), respectively. Differences between the C1s spectra of G and GO are clear in its peak identity. XPS analysis demonstrated that a significant proportion of oxygen in GO existed in the form of –C–OH, these functionalities of oxygen may be associated with hydroxyl and COOH groups (Fig.1(c)). Although major peaks depicted were of C (atomic concentration G=96.54%, GO=63.96%) and O (atomic concentration G=3.41%, GO=35.12%), traces of sulfur were also found in both G and GO. Atomic concentration of S2p increased from 0.05% to 0.92% after oxidation in presence of H2SO4. 3.2.

Surface morphology

SEM micrographs obtained reveals the topography and surface architecture of fabricated membranes (Fig.2). Electrospun PCU membrane shows a fibrous architecture with smooth topography. However, over GOPCU, a very thin transparent film like layer with slight folds/wrinkles could be seen covering the fibrous architecture. These thin folded films appeared after electrospraying the electrospun PCU membrane with dispersed GO nanosheet solution. GO thin film morphology over electrospun PCU fibroporous membrane analyzed from their SEM micrograph presents GOPCU surface, as the PCU membrane wrapped in very thin blanket of GO nanosheets. These thin nanoflakes covering PCU were also analyzed in its freely suspended dispersed state (Fig.2(c)) for its surface area, which was found to be in range of 48±19µm2. 3.3.

Raman spectral study and analysis of electrosprayed GO distribution on GOPCU

Raman spectral analysis was used to get spectral information of thin blanket like structures observed in SEM micrographs. It is an efficient characterization technique for graphene related materials and unlike other techniques detections can be made even for a single layer of material.19 Spectral details of GO, PCU and GOPCU membranes are as shown in Fig.3 which shows distinct peaks of corresponding materials. GO has D band and G band peaks at 1339cm-1 and 1578 cm-1


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respectively while for PCU peak at 1445 cm-1 can be assigned to the symmetric stretching mode of isocyanate (N=C=O) and the bending mode of CH2 .20 Appearance of GO peaks can be observed in spectral details of GOPCU membrane surface also. The GO peaks show a blue shift on GOPCU membrane, wherein D band shifts to 1346 cm-1 and G band has shifted to 1596 cm-1 which may be due to decreased number of layers.21 Thus confirming presence of thin layers of GO on GOPCU membrane. 2D peak which usually appears around 2600-2700cm-1 in Raman spectra for graphene related material and its relative intensity to that of G band is an indication of number of layers.22 Herein absence of any sharp 2D peak with intensity more than that of G band indicates that it may not be a single layer but may be few layers which were seen as transparent coating in SEM analysis. Raman spectral mapping was done to map the distribution of GO over GOPCU surface (Fig.3). The colour red comprises spectral data of PCU and green colour presented that of GO. By analysing thus acquired image using ImageJ software, the colour distribution percentage was measured and coverage of GO was found to be ~ 75% on the PCU membrane surface. 3.4.

Membrane crystallinity and GO aggregation analysis based on XRD diffractograms

Crystallinity is an important property for a polymer membrane, a change in crystallinity of polymer can affect its physical properties23, on the other hand for transferring a thin layer of GO, the coating technique employed may not cause any aggregation of dispersant over the surface to be modified. X-ray diffractograms acquired from XRD analysis of samples may assist in deducing such changes. Powder XRD of GO shows presence of an interlayer spacing of 9.43Å having a peak at 2θ=10.4° as depicted in Fig.4. While diffractogram of GOPCU membrane shows similarity to that of PCU, it was noticed that there were no change in peaks. Neither any new peak appeared, nor did any of the existing peaks disappear, indicating there may not be considerable change in crystallinity of PCU after modification. And the electrospraying technique used to mobilize GO sheets has also prevented an aggregated deposition of GO, that could have given rise to a peak at 2θ=10.4°, as reported earlier.24 Thus thin GO layers transferred on PCU via modified electrospraying method, was found to be an important surface engineering tool that may have wide applications for modification of polymeric membranes. 13 ACS Paragon Plus Environment


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3.5.

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Wetting property

GO thin layers over GOPCU membrane, being an oxidised form of graphene has a potential to attract water molecules due to numerous hydroxyl and epoxide groups on basal plane and carboxyl groups on edges. Water contact angle depicts the wettability of a surface and also one of the major factors affecting the adhesion of any bio-entity on biomaterial surface. A change in hydrophobicity of surface can change the host-material interaction at the surface and can bring about reduction in bacterial adhesion without usage of any antibiotics.25 The PCU fibrous mat showed water contact angle of 121 ± 2°, whereas GOPCU surface embedded with a coating of GO expressed lower values of 92 ± 4° which was closer to 89± 2°, the contact angle exhibited by bulk GO (Table:1). This shows that GOPCU membrane is relatively more hydrophilic compared to that of PCU. A contact angle reduction of ~24% also implies successful transfer of GO over PCU surface and hence exhibiting its inherent hydrophilic property26 over comparatively hydrophobic PCU. 3.6.

Roughness analysis

Folds/wrinkles of thin GO layer wraps, observed on GOPCU might give rise to a rougher surface. Roughness of implant surface is yet another important aspect that affects an implant biocompatibility.27 Roughness values over a surface can change the way it interacts with a cell, more roughness can provide more favorable surface for adhesion due to increased surface area and topography.28 3D topography plot obtained from surface scan during confocal profilometry analysis indicated an increased surface roughness (Fig.5). Further roughness values calculated from confocal profilometry of PCU membrane had Ra = 0.64±0.12 µm, Rq =0.79±0.14 µm and GOPCU membrane had Ra=1.11±0.36 µm, Rq=1.36±0.41 µm. An increment in Ra and Rq values of GOPCU were indicative of the increment in surface roughness that might induce changes in host material interactions. A previous studies has shown that bacterial adhesion over biomaterial may be enhanced by an increase in surface roughness29 while for platelet, it has been reported that the adhesion decreases on the hydrophilic surfaces and increases on the hydrophobic surface.30


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3.7.

Bacterial interaction with GOPCU membrane

Antibacterial property of GO in solution may be attributed to its size

31

, formation of reactive

oxygen species (ROS) by surface functional groups 32, direct contact and presence of impurities like sulfur traces.33 Disc diffusion study is an efficient technique to access antimicrobial property if the antimicrobial agent is capable of leaching out from the disc leading to formation of zone of inhibition (ZOI). The study can be concluded from the presence/absence of zone of inhibition/bacterial clearance around the sample under study. Herein, both PCU and GOPCU tested by agar disc diffusion method showed absence of any ZOI due to lack of any diffusible antibacterial leachant, while the positive control Gentamicin (Gen) disc gave rise to a ZOI of 18mm diameter(d), due to antibiotic leach-out diffusing through agar layer and killing bacterial strain on the plate as seen in Fig.6. Even though XPS data indicated presence of 0.92% sulfur in GO, but this being surface associated was not free to diffuse out. So it was not involved in the mechanism of antibacterial activity, as that by an antibiotic. Hence from the absence of any ZOI it could be inferred that there was no antibacterial leach out which was able to diffuse through agar from the membrane discs. In development of such antibacterial materials it is a serious concern that leaching of antibiotics or antibacterial agents, at some point of time may be suboptimal to kill bacterial strain and might result in formation of drug resistant bacterial strains. Thus, herein the GOPCU membrane has also shown that it has no diffusible antibacterial leach out from the coated membrane. Antibacterial property is exhibited when any material or chemical comes kills the bacteria by contact or by entering it, which then acts by affecting the bacterial metabolism at the level of cell wall synthesis or DNA replication or Translation / transcription during protein synthesis either singly as in the case of the various antibiotics like Gentamicin, Penicillin etc or in combination as in the case of Silver ions. For these prior mentioned agents has to enter the bacterial cell for the effect to manifest itself. It is this phenomenon that is made use in infectious disease treatment while administering antibiotics. When medical devices are used in patients a condition called medical device related infection could result and this occurs when bacteria adhere to the surface of the device, proliferate on it and develop micro colonies resulting in what is commonly referred to as biofilms. In this state the bacteria are impervious to regular antibiotic therapy and biofilms acting as a nidus of infection give rise to chronic infections which can be resolved only by removal of the implant. Biofilm formation is surface associated and a biphasic phenomenon with the initial phase being 15 ACS Paragon Plus Environment


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reversible and guided physical forces like van der Waals forces, gravitational force, Brownian movement etc. the second phase is irreversible and involves chemical interactions and ligand recognition. Hence the current issue raises the need for developing techniques to reduce bacterial adhesion by modifying surface properties or by reducing count of adhered viable bacteria. For understanding this anti-adhesion property of membrane with modified surface bacterial adhesion study was carried out. In the bacterial adhesion study it was found that for GOPCU surface the adhered bacteria after 24h of incubation in culture media was lesser in comparison to bare PCU membrane as shown in ESEM micrograph (Fig.7) and plate count of viable bacterial colony (Fig.8). The percentage adhesion of Gram (+)ve, cocci, Staphylococcus aureus (SA) and Gram (-)ve, bacilli, Pseudomonas aeruginosa (PA) has shown a reduction of 85.5% and 63.5%, respectively (Fig. 8). This analysis has demonstrated that the presence of GO over PCU surface exhibited an efficient bacterial anti-adhesion surface against both Gram positive cocci and Gram negative bacilli type strain of bacteria. Thus, the GO thin films have a wide range of bacterial anti adhesion property against different subtypes and morphology may be due to a combination of surface factors34 like increased hydrophilicity, generation of ROS or traces of sulfur at the direct contact interface of bacteria and GO during dynamic bacterial adhesion study. On the other hand in our study, during the process of electrospraying there may be a reduction in size of dispersed GO nanosheets which was otherwise 48±19µm2, due to shear forces experienced while it passes through the spraying needle and thus as reported in one of previous study done by Perreault et al, decreased surface area of GO may be also involved in inducing some contact based antibacterial properties.31 If medical devices have to reach their true potential of alleviating diseases it should also not give rise to such infections which are commonly called medical device related infections. One of the solutions is surface modification technologies which prevent bacterial adhesion and subsequent biofilm formation. It’s in this context that our work gains significance. The GOPCU does not have any diffusible antibacterial activity as shown by absence of zone of inhibition (Fig.6). But what it does is it prevents bacterial adhesion on to the modified surface vis~ a ~vis the unmodified one (Fig.7).


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3.8.

Interaction of blood components with GOPCU surface

Implanted cardio-device surfaces are always in contact with blood and it is important to ensure that blood cells are not harmed or injured in presence of device inside body. Percentage hemolysis is a crucial part of hemocompatibility and a primary method to ensure absence of any RBC lysis. A slight increment in hemolysis percentage was indicated for GOPCU membrane but the concerned value is still less than 5% (Fig.9). For any biomaterial to be acceptable as a blood contacting device it should have % hemolysis less than 5% as per ISO 10993-4. Hence GO coating has not brought any adverse change in blood contacting property of PCU. Platelet adhesion, activation and aggregation are also considered crucial for hemocompatibility of cardiovascular implants. PCU has been reported to have limited hemocompatibility, due to its inherent surface properties allowing adhesion and aggregation of platelets.35 While in GOPCU, the modified surface showed no aggregation of platelets as visible from ESEM micrograph after qualitative analysis of platelet adhesion using PRP (Fig.10). Quantitative analysis of platelet adhesion was done by exposing samples to both PRP and WB respectively. A reduced platelet adhesion percentage was noticed on GOPCU membrane, as the data graphically represented in Fig.11. Samples exposed to whole blood (WB), PCU membrane showed platelet adhesion of 14.5±0.9% while for GOPCU it was 8.5± 1.5% wherein PCU exceeds acceptable limit of platelet adhesion for a biomaterial to be used in cardiovascular applications that is ≤ 10%. This may be due to a combined effect of alterations in hydrophobicity and roughness, on GOPCU surface. Zingg et al. presented a similar result in his observation that the rougher surfaces brought a reduction in platelet adhesion on the hydrophilic surface and aggravated platelet adhesion on the hydrophobic surface.30 In comparison to PRP exposed samples, a significant increase in platelet adhesion on PCU surface was observed in case of WB (Fig.11), but it was still comparatively lesser on GOPCU. This study exhibit the advantage of surface bound thin GO films in reducing platelet



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adhesion in both PRP and WB environment. Henceforth, promotes application of this surface engineering technique for improving hemocompatible properties of implants. 3.9.

Interaction of mammalian fibroblast cells with GOPCU surface

In case of several implants, mammalian cell adhesion on its surface is an essential property for its long term biocompatibility or functional integration within the site of application.36 GO surfaces have been reported to support adhesion and proliferation of different cell types.37 Herein GOPCU surface having GO thin films offered suitable substrate support for attachment and growth of L929 fibroblast cells. The L-929 cells showed good attachment on PCU and GOPCU fibrous mats, spreading with characteristic morphology and interaction with the surface (Fig.12). Confocal microscopic examination after staining with Hoechst (Fig.12) showed that numerous cells adhered on to GOPCU as on PCU. Actin staining of cytoskeleton (F-actin) of attached cells showed their morphological integrity. Proliferation percentage as analyzed by MTT using L929 showed on an average 82% of survival on PCU and 86% on GOPCU after 24h culture, while a study for extended period of 72 h showed 87% of survival on PCU and 88% on GOPCU (Fig.13). Our results indicate that the modified GOPCU surface doesn’t inhibit cell proliferation and has comparable properties to PCU. The GO-based anti-adhesive surface against bacteria and platelets was found to be biocompatible, allowing active adhesion and proliferation of mammalian fibroblast cells. Hence the novel surface engineering technique developed may be used for modifying biomedical implants and this method can be adopted for other polymeric surfaces for imparting similar properties. 4.

CONCLUSIONS

Herein GO was used as a surface engineering material which has several hydrophilic functional groups like hydroxyl, carboxyl, carbonyl etc. and developed a simple electrospraying based technique to successfully coat GO thin films over a polymeric surface. GO thin films functionalized over PCU have reduced the PCU hydrophobicity by 24%. The modification method used hasn’t considerably affected the crystallinity of PCU nor any aggregation of GO was observed over coated polymeric surface. On the other hand presence of GO immobilized on the surface facilitates the presence of multiple functional groups which are available for further 18 ACS Paragon Plus Environment

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desired modification and functionalization of PCU surface. This surface engineering can hence change an implant surface to have multiple enhanced properties. A single step surface engineering technique using a multi characteristic material like GO nanosheets is capable of conferring differential adhesive & bioactive properties. This is evident from the results that show that this coating imparts bacterial and platelet anti-adhesive property while at the same time promotes adhesion and proliferation of L929 cells. Thus GOPCU proves to be an ideal hemocompatible and biocompatible candidate for medical device development, which prevents bacterial adhesion and biofilm formation. Author information *E - mail: [email protected] Acknowledgement This study was performed at the Sree Chitra Tirunal Institute for Medical Sciences and Technology (SCTIMST) in collaboration with the Indian Institute of Technology Madras (IITM). One of the authors (S.T.) acknowledges the IITM for fellowship and the SCTIMST for facility provided to perform this study. Authors also extend thanks to Mr. Pradeep Kumar at Division of Microbial Technology, SCTIMST for providing technical assistance in carrying out bacterial studies and Mr. Sarath at Amrita Center for Nanosciences & Molecular Medicine, Amrita Institute of Medical Sciences for XPS analysis. References 1.

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Figures with legends

Figure 1: XPS analysis (a) complete spectra of G and GO (b) C1s spectra of G and (c) C1s spectra of GO.

Figure 2: SEM micrograph depicting topography of electrospun PCU (a) and GO sprayed GOPCU membrane (b) (Arrow) shows thin GO flakes, with visible architecture and outline of the electrospun fibers, through it and (c) Dispersed GO flakes(indicated by arrow) coated cover slip. 24 ACS Paragon Plus Environment

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Figure 3: Raman spectra of (a) GO, PCU and GOPCU and (b) mapping of GO thin films (green) distribution over PCU (red) membrane.

Figure 4: XRD diffractograms of GO, PCU and GOPCU.



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Figure 5: 3D plot depicting surface topography of (a) PCU and (b) GOPCU indicating GOPCU having rougher surface.

Figure 6: Zone of inhibition (ZOI) analysis on (a) SA and (b) PA, antibiotic Gentamicin disc (positive control) showed a ZOI (depicted by arrow) with diameter of 18mm, whereas GOPCU and PCU did not show any zone of inhibition.


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Figure 7: ESEM pictures of Gram (+)ve, cocci, Staphylococcus aureus (SA) and Gram (-)ve, bacilli, Pseudomonas aeruginosa (PA) adhered surfaces of PCU and GOPCU, where the adhered bacteria are significantly less.



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Figure 8: Adhesion of Gram (+) ve Staphylococcus aureus and Gram (-)ve Pseudomonas aeruginosa on PCU and GOPCU.

Figure 9: Percentage hemolysis study of PCU and GOPCU surfaces (n=3, P

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