Graphene Oxide Nanocomposite Incorporated Poly(ether imide

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Industrial & Engineering Chemistry Research

Graphene oxide nano-composite incorporated polyetherimide mixed matrix membranes for in-vitro evaluation of its efficacy in blood purification applications

Noel Jacob Kaleekkal a, A. Thanigaivelan a, M. Durga a, R. Girish b, Dipak Rana c, P. Soundararajan b, D. Mohan a,* a

Membrane laboratory, Department of Chemical Engineering, ACT, Anna University, Chennai-600025, India. b Department of Nephrology, Sri Ramachandra University, Porur, Chennai-600116, India. c Department of Chemical and Biological Engineering, University of Ottawa, 161 Louis Pasteur Private, Ottawa, Ontario, K1N 6N5, Canada. *Corresponding Author- [email protected]

Address Dr. D. MOHAN, Ph.D., Professor of Chemistry Membrane Laboratory Department of Chemical Engineering Alagappa College of Technology Anna University, Chennai – 600 025, INDIA. E-mail: [email protected] Telephone Number

: 0091-44-2235-9136

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Abstract Hemodialysis is one of the most commonly used treatments for patients suffering from irrecoverable kidney damage. In our present work we investigate polyetherimide (PEI) mixed matrix membranes (MMMs) as a potential candidate for hemodialysis applications due to its efficient clearance and high biocompatibility. Graphene oxide (GO) was synthesized by the modified Hummers’ method and was then confirmed by X-ray diffraction spectroscopy, Fourier transform infrared spectroscopy, Raman spectroscopy and high resolution transmission electron microscopy. The GO-polyvinylpyrrolidone nano-composite incorporated PEI MMMs were fabricated by a semi-automatic casting unit, using the non-solvent induced phase separation (NIPS) technique. The effect of the nano-composite loading ratio was evaluated by water content; ultrafiltration rate, and porosity; which were all found to increase as the nano-composite content increased. Cross Sectional and top surface morphology was visualized using scanning electron microscopy and atomic force microscopy. The hydrophilicity of these membranes was in consonance with contact angle values. These MMMs demonstrated an increase in biocompatibility – reduced protein adsorption, suppressed platelet adhesion and lower complement activation. Furthermore the prolonged blood clotting time is an indication of the heparin mimic anticoagulant properties of these membranes. The cytocompatibility results by 3(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay and live cell/dead cell staining indicated that there was an increase in cell viability. The membranes with 0.1 wt% GO showed an excellent clearance of the model uremic toxins, namely urea, vitamin B-12 and cytochrome-c in-vitro. The diffusive permeability of these membranes could be comparable to the existing commercial hemodialysis membranes. Thus it can be concluded that these membranes containing a composite of both functional nano-sheets and bioactive polymers have a tremendous potential to be utilized commercially in hemodialysis modules if shown successful in further in-vivo studies with an animal model.

Key Words Graphene Oxide nano-composite, Mixed matrix membranes, polyetherimide, hemodialysis, biocompatibility


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1. Introduction Membrane technology is a necessary component in a number of medical applications, and is crucial in common life-saving treatments. Research on bio-mimicking membranes for biomedical applications such as blood purification, artificial organs and other clinical medical devices are gaining impetus in the biomedical sector 1. With the advancement of research, artificial polymeric materials, such as cellulose acetate (CA) 2, polyacrylonitrile (PAN) 3, polysulfone (PSf) 4, polyethersulfone (PES) 5, polyvinylidene fluoride (PVDF) 6, polyvinyl chloride (PVC) 7, nylon 66 8, polyurethane (PU) 9, and polytetrafluoroethylene (PTFE)

10

, are

being widely applied in various biomedical fields viz. hemodialysis, cardiopulmonary bypass, artificial oxygenators, plasma collection, etc.

Considering the annual increase of about 7-8% in the number of patients that suffer from end stage renal disease (ESRD), hemodialysis membranes are a great area of interest 11. ESRD is defined as an irreversible loss of kidney function where a patient cannot survive without any of the renal replacement therapies (RRTs). Hemodialysis is the most popular and viable of the RRTs wherein accumulated uremic toxins, excess ions and water from the blood are removed and insufficient essential ions are replenished from the dialysate

12

. As for any other device in

contact with blood, the appropriate physical and chemical interface modification is necessary for these membranes in order to achieve a favorable biological response or biocompatibility

13

.

Otherwise, platelet aggregation leads to the formation of thrombus which could possibly cause an artery occlusion, resulting in severe injuries, including heart attacks, and even death 14.

Polyetherimide (PEI) is a novel membrane forming polymer which has been validated for the potential use in biomedical applications

15,16

. PEI is an amber-transparent amorphous

polymer which is thermally, mechanically and chemically stable as well is resistant to many solvents. It is capable of forming flexible thin films which can be attributed to the repeated phenyl and imide groups interspersed with ether linkages and angular bonds.17. However, its suitability for hemodialysis membranes has yet to be investigated. The hydrophobicity of PEI, is similar to commercially available membrane-forming polymers, this causes an insufficient 3 ACS Paragon Plus Environment


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wettability that induces incomplete coating

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, provides for a high adsorption of proteins and

blood cells onto the membrane in any blood contact application.

Various modifications have been carried out to increase the hydrophilicity or biocompatibility. Ran et al. improved the biocompatibility of PES membranes by blending an amphiphilic triblock co-polymer of poly(vinyl pyrrolidone)–b-poly(methyl methacrylate)–bpoly(vinyl pyrrolidone) with it carbon nanotubes whereas

19

. Nie et al.

20

used heparin-mimicking polymer brush grafted

the introduction of heparin-mimicking polyurethane was the

technique chosen by Ma et al. 9. Grafting of zwitterion from polysulfone membrane via surfaceinitiated atom transfer radical polymerization (ATRP) hemocompatible poly(lactic acid)

22

glycol succinate (vitamin E TPGS)

21

, surface zwitterionization of

and the introduction of vitamin E tocopherol polyethylene 23

were few of the other methods chosen to enhance the

biocompatibility of polymeric membranes.

Although each of these modifications imparts unique desired properties to the membrane, blending of polymers with some nano-particles is favored because of the facile fabrication and the resulting permanent modification

24

. Here we have used graphene oxide (GO), an

environmentally and biologically friendlier inorganic nanomaterial in order to improve the properties of the pristine membrane. GO is produced by chemical introduction of oxygen containing groups into pristine graphite material. The epoxide and hydroxyl groups deck the basal plane carbon atoms whereas the carbonyl and carboxyl groups its edge atoms. This ensures that GO is highly hydrophilic and the presence of these groups reduces inter-planar forces, which improves their interfacial interaction

25

. In our study we have incorporated this hydrophilic GO

along with PVP-K90 into the PEI matrix to prepare mixed matrix membranes that synergistically mimic the chemical groups of heparin. Further, introduction of hydrophilic GO maintains the hydrophobic/hydrophilic balance, improves antifouling properties and increases mechanical stability of the membranes 26.


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However, one of the challenges that has to be circumvented when GO is used is to prevent its agglomeration in the polymer matrix. In order to overcome this shortcoming we used an inexpensive, biocompatible polyvinylpyrrolidone (PVP-K90) as a polymeric stabilizer 25. The hereditary hydrophobic carbon skeleton of GO attaches to the polymer matrix via hydrophobic– hydrophobic interactions and π–π stacking, whereas the functional groups (viz. –OH, -COOH, and C=O) gives it the required hydrophilic character and attracts hydrophilic substances by dipole–dipole interaction, hydrogen bonding and dispersion forces 27.

This study aims to produce membranes with a high flux, optimum biocompatibility and efficient removal of uremic toxins, by the incorporation of GO-PVP nano-composite into the PEI matrix forming mixed matrix membranes. Commercially obtained Graphite was chemically oxidized by the modified Hummers’ method and confirmed using X-ray diffraction (XRD), Raman spectroscopy, high resolution transmission electron microscopy (HR-TEM), electron diffraction X-ray (EDX), and Fourier transform infrared (FTIR) spectroscopy. The influence of increasing concentrations of this nano-composite in mixed matrix membranes (MMM) was evaluated in terms of mean pore size, the contact angle and work of adhesion. The morphology was evaluated using SEM and AFM. The mechanical stability of the modified membranes was evaluated in terms of tensile strength. Blood compatibility was evaluated with plasma protein adsorption, platelet adhesion, plasma recalcification time; complement activation, activated partial thromboplastin time (aPTT) and prothrombin (PT). Furthermore, the cytocompatibility was evaluated by cell culture (hepatocyte cell line), live/dead cell imaging and 3-(4, 5-dimethyl2-thiazolyl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay.

2. Materials and Methods 2.1 Materials Commercial grade polymers polyetherimide (PEI) from Sigma Aldrich Inc., St. Louis, MO (melt index 9 g/10 min) was used after being dried in a vacuum oven at 80°C for 48 h. The solvent Nmethyl-2-pyrrolidone (NMP) from SRL Chemicals Ltd., Mumbai, India was dried using 4Å 5 ACS Paragon Plus Environment


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sieves. Molecular grade Urea (MW 60.06 Da), Vitamin B-12 (MW 1355 Da), Cytochrome-c (MW 11.8 kDa) and Bovine serum albumin (MW 64 kDa) purchased from Himedia Laboratories Pvt. Ltd., Mumbai, India, were used in the solute diffusion studies. Urea Nitrogen (Diacetyl) reagents from Erba Mannheim, Mnnheim, Germany, were used for the determination of the solute concentration. Graphite (fine powder 98%) extra pure, sodium nitrate, hydrogen peroxide and potassium permanganate were obtained from Loba Chemie Pvt. Ltd., Mumbai, India. These materials were used as is. Milli-Q water was used for all solution preparations and studies.

2.2 Preparation of Graphene Oxide 3 g of graphite powder was added to a round bottom flask containing 70 mL conc. H2SO4 and stirred in an ice bath. To this mixture 9 g of KMnO4 was added slowly to ensure that the temperature was maintained lower than 15°C. After half an hour, the flask was transferred into a 40°C bath and vigorously stirred for 1 h. Then 150 mL of water was added drop wise and the solution was maintained at 95°C for 1 h. The solution changed color from dark brown to yellow when an excess 500 mL of water and 15 mL H2O2 (30%) was added. This solution was allowed to settle and the solids were washed with dilute HCl (250 mL) to remove metal ions

28

. The

solution was filtered and centrifuged in hot conditions to remove the precipitates soluble in warm water. This yellowish brown residue was then ultrasonicated to get fully exfoliated graphene oxide. Next it was dried in a vacuum oven overnight and then used for further characterization and membrane preparation.

2.3 Characterization of GO 1) Wide-angle X-ray scattering (WAXS): the X-ray powder diffraction patterns were recorded on a Bruker-AXS diffractometer (Bruker, Germany) using Cu Kα radiation (λ = 0.154 nm).

2) Confocal Raman Spectroscopy: Raman measurements were performed on an In Via/Reflex Laser Micro-Raman spectroscopy (Renishaw, Gloucestershire, England) with 6 ACS Paragon Plus Environment

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an excitation laser beam that had a wavelength of 514 nm. The powders of graphite and graphene oxide were then placed on a clean SiO2/Si substrate that was used for the Raman measurement.

3) HR-TEM with EDX Microscopy: Transmission electron microscopy (TEM) images (including high resolution images), selected area electron diffraction (SAED) patterns and electron diffraction X-ray (EDX) were acquired using a JEM-3010 TEM (Jeol Ltd., Tokyo, Japan) operated at 200 kV. Freshly sonicated graphene oxide (～0.02 mg/mL) was spread on carbon-coated copper grids, and blotted after 30 s.

4) FT-IR Spectroscopy: Powdered samples were spread on the attenuated total reflection (ATR) crystal and percent transmittance between 400 cm−1 and 4000 cm−1 was measured on a spectrophotometer (Nicolet Avatar 370, Thermo Electron Corp., Madison, WI)

5) UV-Visible Spectroscopy: The absorption spectra were recorded on a UV-vis spectrometer (Libra S50, Biochrom Ltd., Cambridge, England). The aqueous suspensions of graphene oxide were used as the UV-vis samples, along with pure water taken as reference.

2.4 Preparation of the mixed matrix membranes. Initially GO (0, 0.025, 0.050, 0.1 and 0.2 wt%) was sonicated in the solvent (NMP) for 1 h, after which 2 wt% PVP-K90 was added and further homogenized by vigorous stirring for 2 h. To this, 16 wt% PEI was added and a homogeneous dope solution was prepared by mechanical stirring for 24 h. The dope solution was then degassed by applying a vacuum. A semi- automatic casting unit was used to prepare these mixed matrix membranes. The dope solution was casted on a dust free glass plate maintaining a thickness of 200 µm at a relative humidity (RH) of 25%. The glass plate was then immediately immersed in a water bath containing 0.2% solvent. The nascent membranes were washed with warm water (35±2°C) and then stored in distilled water until further testing. 7 ACS Paragon Plus Environment


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2.5 Porosity and Average pore size. Membrane porosity (Ɛ %) was measured by the gravimetric method. The membrane porosity is obtained by:

⁄

Ɛ % =

/

(1)

Where; Ww and Wd are the weights (g) of the wet and dry membrane, respectively, Dw and Dp are densities (g/cm3) of the water and polymer membrane, respectively. These tests were replicated thrice and their average value has been reported.

The average pore radius rm (m) is determined by the filtration velocity method, using the Guerout-Elford–Ferry equation 29:

=

. .× ×!×"

(2)

where, η is the water viscosity at 25°C (Pa s), l is the membrane thickness (m), Q is the water flux (m3/s), A is the membrane area used for filtration (m2), and P is the predetermined operating pressure (Pa).

2.6 Fourier transform infrared spectra and surface streaming potential Attenuated total reflection-Fourier transform infrared spectra (FTIR/ATR) for prepared MMMs were compared to the pristine membrane using a spectrophotometer (Nicolet Avatar 370, 8 ACS Paragon Plus Environment

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Thermo Electron Corp., Madison, WI). The measurement of the surface streaming potential was determined using a surface analyzer (SurPass, Anton Paar, Austria).

2.7 Contact angle and work of adhesion The static contact angle (CA) is a measure of the surface hydrophilicity of the membrane. The CA of the prepared membranes was obtained by a contact angle goniometer (OCA15, DataPhysics, Filderstadt, Germany) equipped with video capture. 5 µL double distilled water was carefully placed on the 2 × 2 cm membrane sample which was affixed on a glass slide. The reading was taken after 10 s. The measurement was done on five different locations of the membrane surface to get reliable data.

The work of adhesion (W) explains the interactive forces between liquid and solid surfaces. This was calculated using the Young-Dupree equation:

# = $% 1 + ()*+ (3)

Where, γL is the surface tension of the water (72 mJ) and θ is the contact angle 30.

2.8 Morphological analysis The cross sectional and top surface morphologies of all prepared membranes were determined by SEM and AFM respectively. A 1 cm2 sample of the membrane was dried using a series of ethanol solutions, then fractured in liquid nitrogen and then loaded onto the scanning electron microscope (FEI Quanta-400 FEG) to observe the cross sectional morphology.



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Atomic force microscopy in the non-contact mode analyzed the surface morphology and roughness of the membranes. The AFM apparatus used was a XE-100, Park Systems Corp., Suwon, South Korea. The prepared membranes were cut into small squares (1 cm2) and affixed onto the metal substrate. The roughness values reported are the average of three different scan areas of 5 µm x 5 µm each.

2.9 Ultrafiltration rate (UFR) In dialysis practice, to evaluate and control the performance, the ultrafiltration rate is evaluated by using the following equation:

01

7

UFR /2 3 00456 = ".8.9

(4)

Where V is the quantity of permeate collected in mL, A is the membrane surface area (m2), and P is the operating pressure (mmHg), t is the time for which permeate is collected (h).

2.10 BSA retention and Anti-fouling It is imperative that the membrane should prevent loss of albumin from the blood stream during dialysis. The BSA concentrations in the original (Co) and the corresponding final mimic blood (Cf) were determined with a UV–vis spectrophotometer (Libra S50, Biochrom Ltd., Cambridge, England) at a wavelength of 280 nm and calculated utilizing a standard curve. The BSA retention (R) of the membrane was then calculated by:

;

:% = /;< 6 × 100 (5) =


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Three different membrane areas are used and the retention is given as the standard deviation of the same. The fouling-resistant ability was evaluated by the measurement of the time-dependent fluxes of PBS followed by that of BSA solutions. Then the membranes were cleaned using water for 20 min and both fluxes were measured again. The flux recovery ratio (FRR) was calculated using the following expression 31. Total of 3 such runs were carried out.

A

?::% = @ B D × 100 AC

(6)

Where J0 and Ji are solution fluxes for the ith run before and after protein solution ultrafiltration, respectively.

2.11 Bio-compatibility Characteristics 2.11.1 Protein Adsorption 1 x 1 cm membrane samples were equilibrated in a phosphate buffer solution (PBS) for 12 h before use. Then, these samples were incubated in 1.0 mg/mL or 0.05 mg/mL protein (BSA or FnG respectively) solution at 37°C for 2 h and subsequently rinsed with PBS solution followed by double distilled water. Following this, the membranes were then immersed in 2 mL of 2 wt.% sodium dodecyl sulfate (SDS) aqueous solution at 37°C for 1 h to remove the adsorbed protein. The Micro BCA™ Protein Assay Kit (Thermo Scientific) was used to determine the protein concentration in the SDS solution, which is the quantity of adsorbed protein. The experiment was carried out in triplicate 9.

2.11.2 Platelet adhesion Healthy fresh human blood (26 years, male) was collected using sodium citrate lined vacutainer. Here the ratio of the anticoagulant to blood was 1:9 v/v. The blood was centrifuged at 1000 11 ACS Paragon Plus Environment


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r.p.m. for 15 min to obtain platelet-rich plasma (PRP). The membrane was then immersed in a PBS solution and equilibrated at 37°C for 1 h. The PBS was then removed and 0.5 mL of fresh PRP was added. Lastly they were then incubated at a 5% CO2 atmosphere at 37°C for 1 h.

The lactate dehydrogenase (LDH) method was used to determine the number of platelets adhered to the samples. The concentration of adhered platelets were calculated from the calibration plot 32. Here a linear relationship was obtained between the LDH activity of aliquots and the known concentration of platelets was counted using a hemocytometer. Another set of these membranes; the platelet adhesion was evaluated using SEM. After contact with the PRP the membranes were fixed using glutaraldehyde in PBS. The samples were dried using the solvent exchange method. The platelet adhesion was observed using a SEM (FEI Quanta-400 FEG) 33.

2.11.3 Thrombin–antithrombin III (TAT) generation The membrane (1 cm2) was incubated with 1 mL of healthy human fresh whole blood (man, 26 years old, sodium citrate anticoagulant) at 37°C for 1 h, followed by centrifugation at 4000 r.p.m. for 15 min to obtain the platelet-poor plasma (PPP). TAT was measured by an enzymelinked immunosorbent assay (ELISA) with commercially available kits, Enzygnost TAT micro (USA) 20. Duplicate runs were carried out to ensure reliability.

2.11.4 Clotting time Activated partial thromboplastin times (aPTTs) and prothrombin times (PTs) for the membranes were measured by a semi-automated blood coagulation analyzer (CA- 50, Sysmex Corp., Kobe, Japan) in order to evaluate the contact activation pathway and the abnormality in the conversion of fibrinogen to fibrin in coagulation, respectively. Here the membrane sample discs of 0.5 cm2 were incubated in 0.20 mL PPP at 37°C for 30 min, and then the aPTT and PT were measured. In order to get a reliable value, the test was repeated three times for each sample23. 12 ACS Paragon Plus Environment

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2.11.5 Plasma recalcification time (PRT) Membrane discs of 0.5 cm2 were immersed in the PBS solution and equilibrated at 37°C for 1 h. 100 µL PPP was placed on the sample film attached to a glass slide, and incubated statically at 37°C; and then 100 µL of 25 mM CaCl2 aqueous solution was added to the PPP. The plasma solution was then monitored for clotting by a UV-Vis spectrophotometer. In order to get a reliable value, the test was repeated three times for each sample.

2.11.6 Complement Activation 20 mL of human blood was drawn from healthy adult volunteers without the addition of any anticoagulant. The collected blood was incubated for one hour at 37ºC. The resulting clotted blood was then centrifuged at 2000 rpm for 20 min in order to separate the serum. Membranes (1×1 cm2) were immersed in 2 mL of serum and further incubated at 37ºC for 1 h. The concentrations of complement components, C3a and C5a, left in the serum after incubation with membranes were determined with the Human Elisa kit for C3a and C5a (Assay Pro, USA) respectively.

2.12 Toxic Solute removal The efficiency for the removal of uremic toxins and other middle molecular weight solutes were tested in a single stage test cell in a counter current manner. The membranes having an area of 38 cm2 were placed between the solute and dialysate chamber, each having a volume of 10 mL. The solutes chosen were urea (60 Da), vitamin B-12 (1355 Da) and cytochrome-c (~12 kDa). Freshly prepared PBS solution was used in the dialysate side. Constant flow rates of 50 mL/min and 100 mL/min were maintained for solute and dialysate reservoirs correspondingly. The samples were drawn at fixed intervals and were then analyzed. The concentration of urea was determined using an Erba Mannheim (Urea BUN) kit; the concentrations of vitamin B-12 and cytochrome-c were directly measured using a UV-Visible spectrophotometer at wavelengths 360 nm and 550 nm receptively. Three runs were carried out for each solute to ensure the consistency of performance for the membranes. 13 ACS Paragon Plus Environment


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The overall mass transfer coefficient K0 is determined by the solute component mass balance between the two chambers.

EF =

GH/

∆;J K∆; 6 2

J J O P× 92 9J MN M

8× L

(7)

Where t1 and t2 are the sampling times, ∆C is the difference between the solute concentrations of both cells at each sampling time, Vb and Vd are the solution volumes in each cell (Vb=100 cm3, Vd=200 cm3) and S is the effective membrane area (38 cm2).

The overall resistance to mass transfer is comprised of two components- the bulk fluid resistance and the membrane resistance as given by equation 8:

QC

=R

STS

+U

N

(8)

The factor ‘2’ in the above equation accounts for the presence of the boundary layer in both of the chambers. The bulk mass transfer coefficient kb was evaluated using equation 9:

UN V W

= X :Y F.Z [( F.\\

(9)

Here r is the radius of the reservoirs, D∞, is the diffusion of the solute at 25°C, Re is the Reynolds number, and Sc is the Schmidt number. The parameter α depends on geometry and was 14 ACS Paragon Plus Environment

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determined to be 0.26

34

. From these equations the diffusive permeability of the solute through

the membrane (pmem) can be determined.

2.13 Cell Cytocompatibility 2.13.1 Cell Culture Hepatocytes were co-cultured from Chang Liver cell line with supplementation of Dulbecco Eagle’s Minimum essential medium (MEM) and 10% fetal bovine serum. Hepatocytes were isolated by a two-step gradient centrifugation, and phosphate buffered saline (PBS) was used to wash the cells, and then finally diluted to 104 cells/µL and the viability was then confirmed by tryphan blue.

2.13.2 MTT assay Membranes of equal sizes (1 cm2) were cut, equilibrated in PBS and sterilized under UV light overnight. These cultured cells were seeded on to 24 well culture plates at an equal density of 104 cells/µL and incubated for 24 h at 37°C in a 5% CO2 incubator till the cells attained the required morphology. The membranes were then incubated on to the cells and incubated in a CO2 incubator for 24 h at 37°C and cell viability was determined by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay. Cells cultures in wells without membranes served as control 5. MTT was prepared at a concentration of 1 mg/mL and 50 µL was added to each well and incubated for 4 h at 37°C in 5% CO2. Mitochondrial dehydrogenases of viable cells cleaved selectively to the tetrazolium ring. Then 50 µL of DMSO was added to dissolve the formazan crystals, which yields a purple color. The level of the reduction of MTT into a colored formazan salt by the living cells was measured spectrophotometrically at 570 nm using a micro plate reader (Perkin Elmer ENFIRE, multimode reader). Assay was performed in triplicates and the statistical significance was determined by the student t-test with the level of significance at P < 0.05.



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2.13.3 Cell Viability using Acridine orange/Ethidium bromide (AO/EtBr) staining For cell staining, the membranes and medium were removed through following the incubation of membrane in seeded cells at 37°C in the CO2 incubator. Cells were then stained with 1mL of acridine orange / ethidium bromide prepared in PBS and incubated for 5 min. Acridine orange cleaves the membrane and stains the intact viable cells with the color green while ethidium bromide, which is membrane impermeable, stains the apoptotic cells in red. Fluorescent stained cells were viewed under a fluorescent microscope and images were captured with a Nikon camera built in the microscope.

3. Results and Discussion 3.1 Characterization of GO We have employed the modified Hummers’ method for the synthesis of GO from graphite using sulphuric acid and KMnO4 as oxidizing agents

28,35

. The Fourier transform infrared (FTIR)

spectra (Fig. 1A) revealed the existence of OH stretching vibrations (3400 cm-1), stretching vibrations from C=O (1720 cm-1), C=C skeletal vibrations from unoxidized graphitic domains (1640 cm-1), C-OH (1224 cm-1), and C-O (1050 cm-1) functional groups, suggesting that oxygencontaining groups are introduced into the graphene. Disorder of structures or site defects of G or GO are identified and characterized using the powerful Raman spectroscopy technique. The Raman spectrum as seen in Fig. 1B displayed a fortify peak assigned to the vibration of sp2banded carbon atoms at 1580 cm-1 (G band) and another strong peak assigned to the vibration of disordered sp3 carbon at 1350 cm-1 (D band)

36

. The D peak arises due to first-order zone

boundary phonons which is absent in pristine graphite. Fig. 2A shows a TEM image of exfoliated graphene oxide sheets. It shows a number of stacked GO nano-sheets wherein each of the single sheets appears transparent and are folded over or curled on one edge. EDX analysis gave a C:O ratio of 3:1. The 2Ɵ peak seen from the diffraction analysis is seen to be centered at 11.36° (Fig. 2B), corresponds to the diffraction from the (002) plane, which indicates an interplanar spacing of 7.78 Å compared to 3.4 Å for the pristine graphite material. This reveals the intercalation of oxygen functionalities into the ordered graphite structure which facilitates the hydration and exfoliation of GO sheets in aqueous media

37

. The spectra are plotted in the 16

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wavelength range from 200 to 500 nm for both water and NMP. In the case of NMP the data is plotted from > 260 nm as a result of the impossibility of properly compensating for the strong absorption of the solvent at smaller wavelengths. The π–π transitions of the doubly bonded aromatic carbon atoms38 present on the GO backbone, is responsible for the highest absorption peak at 231 nm(Fig. 2C) observed from the UV spectra. Further from this spectra at 300nm a shoulder peak observed be attributed to n→π* transitions of the C=O moieties. These results are comparable to earlier studies.

3.2 Preparation of PEI/GO mixed matrix membranes Pre-weighed quantity of the dried GO was sonicated with the PVP to form a nano-composite. These nano-composites were incorporated into the polymer dope (PEI + NMP) solution by physical blending. The mixed matrix membranes were prepared by first casting the solution with a preset thickness using a semi-automatic casting unit followed by the NIPS technique. The optimized concentration of PEI and PVP were found to be 16 and 2 wt%, respectively. M0 indicates the pristine membrane without any GO, where M1, M2, M3 and M4 are membranes with increasing concentrations of GO (0.025, 0.05, 0.1 and 0.2 wt%, respectively). The coagulation bath contained distilled water with 0.2% w/v NMP, and the bath temperature was maintained at 20°C. After 2 h the nascent membranes were transferred to a bath containing warm water and was stored overnight. Furthermore no elution of GO was observed into the bath. These membranes were washed and stored until further use. It can be seen that the surface of the membranes becomes darker as the GO loading increases as seen in Fig. 3.

3.3 Surface chemical structure and surface streaming potential The ATR-FTIR spectroscopy was used to analyze the surface enrichment of GO on the membranes. Infrared spectrums of the MMMs are shown in Fig. 4. For the pristine membrane the bands at 1778 and 1718 cm-1 correspond to the asymmetric and symmetric stretching vibrations of the carbonyl, respectively. The absorption band at 1350 cm-1 is assigned to the bond stretching vibration of C–N in phthalimide rings. The aryl ether bonds is confirmed by the vibrations at 17 ACS Paragon Plus Environment


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1270, 1232, 1072, and 1018 cm-1

39

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. In comparison with pristine PEI membrane, the GO

modified membranes exhibited intense and wider peaks at ~3400 cm-1, which indicated that the surface hydrophilicity was obviously improved due to the resident GO on the top surface. Similar observations were also made for GO incorporated PVDF membranes 40. The pristine M0 membrane showed a surface charge of -18.20 mV which increased to -30.60 mV for membrane M3 at a pH of ~7.2. This was done using KI solution and was then adjusted for pH. The decrease in surface charge is an indication of GO nanocomposites being oriented at the membranes surface which is in accordance with the optical images of the MMMs.

3.4 Morphology Analysis The micro-structure cross sectional morphologies of the pure and mixed matrix membranes were analyzed using SEM as seen in Fig. 5. All of the membranes had an asymmetric structure composing of dense skin layers and porous sub-structure. Both pore size and pore numbers have notably increased with increasing concentration of GO. And it is seen that the skin layer thickness decreases with an increase in GO concentration which in turn, explains the pure water flux. In the composite membranes, the amphiphilic GO-PVP nano-composite migrated and were concentrated on the membrane surfaces. Consequently it can be hypothesized that the increase in pore size and relatively compact architecture of the mixed matrix membranes would reduce the time required for removal of the toxins as the flow rate, which is a limiting factor, increases. The presence of GO makes the system thermodynamically unstable, leading to a quicker L-L phase separation with a more porous structure 41.

The use of biomaterials in applications where they come in contact with the blood cells or other tissues could lead to a cascade of biological responses originating with an a protein adsorption (viz fibrinogen), which is followed by attachment and activation of serum platelets and also cell cytotoxicity20. The top surface morphology of the MMMs are as shown from AFM analysis seen in Fig. 6. From the topography it can be understood that as the GO nano-composite concentration in the MMMs increased, the surface roughness would also increase. The light and dark regions seen from the AFM mapping correspond to the high and low (membrane pores) 18 ACS Paragon Plus Environment

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areas of the membrane sample. The Ra, Rq and Rz parameters given in Table 1 are the average values of three 5 µm x 5 µm areas scanned. This possibly demonstrates that the hydrophilic nature of GO leads to a faster exchange of S-NS during the NIPS process. The rate of solvent exchange increases which accounts for the spheres or nodules of polymer on the top surface which accounts for the greater roughness. This phenomenon could explain the increase in porosity and has been observed by other researchers

42,43

. However the surface roughness of all

the MMMs were lower than the pristine PEI membrane. So it can be seen that the incorporation of GO caused the huge ‘‘peaks’’ and ‘‘valleys’’ to be replaced by numerous small ones, which led to an overall smoother membrane surface. The surface roughness parameters are as shown in Table 1.

3.5 Contact angle and Work of Adhesion The water contact angle measurements can analyze the hydrophilicity of the membrane surface. A hydrophilic surface will exhibit a smaller contact angle as it permits the spreading of water on its surface. Fig. 7 shows sessile drop contact angle of the fabricated MMMs. As shown, a significant decline of the contact angle is observed when a greater amount of GO was incorporated into the membrane matrix. During the vitrification of the MMMs, in the course of phase inversion the nano-composites migrate spontaneously to the water interface in order to reduce the interfacial free energy

44

. The darker color on the top surface of the membranes in

comparison to that at the bottom indicates a migration to the membrane surface. The large amount of –OH and –COOH groups of the GO orient themselves in a way to impart their hydrophilic properties to the membrane whereby increasing the adsorption of water and, therefore, improving the membrane water permeability. The adhesion on hydrophobic surfaces is greater compared to surfaces possessing high concentration of polar groups. Lower interfacial free energy surface corresponds to lower fouling in most membrane separation processes. In the MMMs, in order to lower the surface free energy of the system; during the membrane formation lower surface energy nanoparticles preferentially migrate to the top surface thereby creating a gradient between the top surface and the bulk of the membrane45,46. Thus the most promising alternative in preventing protein or platelet adsorption is using materials that have a low surface energy and low adhesive surface. 19 ACS Paragon Plus Environment


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3.6 Porosity and Pore Size (Filtration Porosity Method) The effects of the GO content on the porosity and mean pore size were also listed in Table 1. Both porosity and mean pore size of membranes had increased with the increased addition of GO, which was consistent with the results of SEM. The presence of GO nano-composite in the dope solution caused micro phase separations which facilitated the generation of a polymer poor phase, thus suggesting that the existence of GO is beneficial for membranes that have a high porosity and mean pore size 40. The pristine M0 had a pore size of 32nm, which increased to 76 nm for membrane M4.

3.7 BSA retention Renal failure patients had an occurrence of albumin loss associated syndrome. Albumin is approximately 67 kDa and is lost during the hemodialysis. Therefore, the ideal membrane should avoid albumin loss during treatment. All of the prepared membranes were subjected to a protein retention study. They showed a retention ratio greater than 95% for the BSA protein. An increase in the GO concentration (beyond 0.1%) however, it shows a small loss of BSA to the dialysate (Table 2). M4 shows a retention of only 95% which could be explained by the larger pores as visualized in the SEM, or due to the presence of micro cracks on the skin layer due to the high concentration of GO.

3.8 Ultrafiltration rate and antifouling properties During ultrafiltration, the permeability of a membrane is of pronounced importance. Membranes with excellent antifouling and reasonable protein rejection ratios are favorable in hemodialysis. The UFR of the pristine membrane was observed to be 6.8 mL/h·m2·mmHg and the most efficient membrane M3 had a UFR of 26.8 mL/h·m2·mmHg. The antifouling properties could be characterized in terms of flux recovery ratio during the ultrafiltration of the protein solution. It 20 ACS Paragon Plus Environment

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can be seen that membrane M0 possessed very low flux recovery owing to its inherently hydrophobic nature. However, even a small amount of the nano-composites showed significant improvement in FRR values as seen in Table 2. The improvement of PBS flux and a small decline in BSA rejection ratio could be attributed to the following reasons: 1) the addition of GO causes a formation of a larger number of interconnected pores with greater size, 2) the existence of micro surface defects due to aggregation of the more hydrophilic GO onto the surface.

3.10 Biocompatibility Studies 3.10.1 Protein Adsorption A key parameter in evaluating blood compatibility is the amount of protein adsorbed onto the membrane surface and this is one of the main initial events that leads to blood clotting

47

.

Numerous factors influence the protein adsorption onto the membrane surface- protein size, shape, temperature, pH, surface charge, hydrophilicity/hydrophobicity, and surface topology. Other factors in consideration are co-adsorption of low-molecular-weight ions, the strength of functional groups, intermolecular forces between the adsorbed molecules, the composition of the protein solution, and the chemistry of the surface

48

. The adsorption of protein fibrinogen in

blood plasma is important since it paves the way for platelet adhesion so that it can bind to the platelet GP IIb/IIIa receptor 49. Apart from FnG many other plasma proteins, including albumin and gamma- globulin, are also adsorbed onto hemodialysis membranes. From protein adsorption studies, the protein concentrations were formulated to resemble physiological concentrations, so that the concentration of albumin used would be much greater than the fibrinogen. As shown in Fig. 8, the pristine PEI membrane (M0) has shown the highest protein adsorption of both BSA and FnG (30.6 and 17.4 µg/cm2 respectively). It can be seen that the incorporation of GO into the membrane matrix results in lower protein adsorption. The membrane M4 shows the highest resistance to protein adsorption, which can be explained by the increase in hydrophilicity and the lower surface roughness. BSA protein, at a pH of 7.4 is negatively charged so it has a lower adsorption tendency on more negative surfaces because of charge repulsion50.



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Generally, membranes are prepared using the NIPS technique and the hydrophilic components (here GO) preferentially migrates to the solution-membrane interface due to the low interfacial energy between the water and hydrophilic components. The GO present on membrane surfaces extends its functional group into the surrounding aqueous environment, and hydrogen bonds could be formed between water and the –OH/-COOH groups present on the GO sheets. This forms a hydration layer and due to the effect of steric exclusion, protein adsorption could be inhibited

51

. The amounts of protein adsorption that were reported in previous publications

showed a significant difference, from not more than 1.0 µg/cm2 to several hundreds of µg/cm2. Due to modifications where hydrophilic groups are introduced onto the membrane surface, a sharp decline has been observed by other researchers

52–54

. Further, it was explained that the

protein adsorption on hydrophilic surfaces occurred in one of the two modes. Where one is that the surface is aggressively covered by the protein it comes in contact with, and the other involves an inhibition of adsorption by the initially deposited layer to prevent further adsorption (surface passivation) 5.

3.10.2 Platelet Adhesion Another vital tool for the appraisal of blood compatibility is platelet adhesion. Any foreign material that is in contact with blood initially adsorbs the blood plasma proteins. This is followed by platelet adhesion and activation of the platelets, which in turn induces thrombus formation 55,56

. A crucial step in the process of thrombus formation is the extent of platelet adhesion and

platelet aggregation. It was observed that the platelets adhering on the pristine PEI membrane were aggregated in large numbers on the membrane surface and possessed a network like structure with deformed and extended pseudopodium. However in the modified MMMs, the platelets retained their original shape without deformation and their numbers were fewer. We also noticed that the conformational change of the platelet was suppressed even if it adsorbed onto the membrane surface. The amounts sharply declined with an increase in percentage of GO as seen in Fig. 9. It is understood that the fibrinogen is a known mediator for platelet adhesion and aggregation

57

. The platelet adhesion results were consistent with the previous FnG

adsorption results. The results from SEM were also correlated with those obtained by LDH Assay. The number of platelets adhered decreased from 22 x 104 cells/cm2 for pristine PEI 22 ACS Paragon Plus Environment

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membrane to almost 5 x 103 cells/cm2 for M4 membrane. Other researchers have also shown that on inclusion of GO or GO-g-pMPC, platelet adhesion is suppressed 58. 3.10.3. TAT generation The adhered platelets have different types of interactions with various coagulation factors, one of them- the coagulation product thrombin is known to be a potent platelet activating agonist. The platelet activation level could be reflected by the platelet factor 4 (PF4) expressed by the activated platelets; and the thrombin that resulted from the activation of coagulation cascade combined with antithrombin III to generate thrombin–antithrombin III (TAT) complexes and, in effect, demonstrated the extent of thrombin generation 59,60.

The level of thrombin generation in the plasma after it had contact with blood, was also measured by Thrombin–antithrombin (TAT) complexes. These complexes are formed following the neutralization of thrombin by antithrombin III, and are used as a surrogate marker for thrombin generation 61. Thrombin generation kinetics are measured by the appearance of plasma TAT complexes, demonstrating the anticoagulant property of the GO, when incorporated into the membrane matrix. It was observed that the TAT concentration for the M0 membrane remained higher than the plasma (control) values (Fig. 10). M1 and M2 which had lower concentrations of GO also displayed significant TAT generation. Membranes M3 and M4 showed suppressed generation of TAT complexes. It is assumed that an augmentation of GO concentration increased the hydrophilicity of the membrane surface and this, in turn, led to a lower thrombus generation. The results determined that the modified membranes obviously showed lower platelet activation and the minimal coagulation cascade activation when compared with those of pristine PEI membrane.

3.10.4 Clotting time When blood is made to flow through any extra corporeal circuit, it comes in contact with artificial surfaces, and the hemostatic system may respond in diverse ways depending on the 23 ACS Paragon Plus Environment


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nature and the duration of stimuli. These surfaces will rapidly adsorb plasma proteins at the blood-material interface which will mediate subsequent thrombotic events and clotting. The aPTT and PT are principally used to examine mainly the contact activation pathways and common coagulation pathways of blood coagulation factors

62

. Activated partial thromboplastin

time (aPTT), the global screening procedures used to evaluate coagulation abnormalities in the intrinsic pathway, can also be used to detect functional deficiencies in Factors II, III, V, VIII, X, or fibrinogen 63. Clotting times of the modified membranes was seen to have improved compared to the pristine membrane as seen in Fig. 11. The aPTT of membrane M-4 was found to be twice as much as M-0. Moreover the PT was also slightly increased with the addition of GO into the polymer matrix. M-4 has an aPTT of 75 s which demonstrates that there is a delay in clotting. These results were in accordance with other researchers who introduced functionalized MWCNTs into the polymer matrix 20.

Blood clotting time was considerably improved in PEI mixed matrix membranes. The result might have partial contribution from its surface hydrophilicity since this results in less protein adsorption and platelet adhesion on the membrane surface. Thrombosis is usually initiated by the adsorption of plasma proteins, followed by the adhesion of platelets. The high hydrophilic surface formed on the PEI mixed matrix surface extended clotting time, lowered protein adsorption, suppressed platelet adhesion and activation.

3.10.5 PRT In the interaction of blood with negatively charged surfaces the intrinsic pathway is triggered by surface contact and this in turn sets off a cascade of reactions requiring Ca2+. Plasma recalcification profiles serve as a measure of the intrinsic coagulation system 64. The presence of turbidity is an indication of clot formation. Citrated platelet poor plasma (without the addition of CaCl2) serves as a negative control, since it should not form a clot.


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The PRT basically has the same clinical significance as the whole blood coagulation time (CT). Though it is generally more sensitive and accurate than CT, and is comparable to the in vivo experiments 65. The PRT of M0 is similar to the positive control values. On increasing the GO content the PRT is found to increase in accordance with the aPTT values as observed from Fig 12. 3.10.6 Complement Activation Complement activation is the body’s defense mechanism against pathogens or any “non-self” entities. The human complement system consists of 20 or more different plasma proteins which function either as enzymes or binding proteins. Complement activation is initiated by classical or alternative pathways where the terminal pathway is common to both. Both pathways contain an initial enzyme that catalyzes the formation of the C3 convertase, which consecutively generates the C5 convertase allowing the assembly of the terminal complement complex

66

. Complement

activation triggers the host defense mechanism by generating the localized inflammatory mediator. This could be quantified by the measurement of the produced anaphylatoxins C3a, C4a, and C5a. We have determined the concentration of C3a and C5a using the ELISA assay for the complement activation study. The assay was also used as a way to characterize the bloodmembrane compatibility of the GO incorporated MMMs.

Both markers of complement activation C3a and C5a are elevated in M0 manifesting the generation of acute inflammatory response of the pristine membranes. C3a and C5a concentrations were 42 ng/mL and 1.6 ng/mL, respectively (Fig. 13). The membranes loaded with GO tend to inhibit the complement activation and the membrane M4 exhibited the least ability to activate the complement system. These detailed studies might provide useful information to other studies on material with negatively charged surfaces and extend the applications of the “heparin-like modification method”.

3.11 Solute Permeation through the MMMs



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In hemodialysis, the solutes with low molecular weights are removed from the solution by allowing them to diffuse into a region of low concentration. Hence for the MMMs the permeability coefficients of the solutes are in concordance with their molecular weightpermeability coefficient of urea > vitamin B-12 > cytochrome-c. Fig. 14 shows the permeability coefficients of the chosen solutes urea, vitamin B-12 and cytochrome–c. Urea is a standard solute marker and has a high clearance during hemodialysis. Its movement in and out of the RBC’s is rapid. The dialysis clearance of urea is greater than the plasma flow. Therefore, the clearance of urea is higher than other solutes. Some researchers hypothesize that membrane solute clearance is not directly correlated with its ultrafiltration rate

67

; however our results do indicate a

68

correlation between the two as seen by Gao et al. . Though the permeability coefficient of urea through the membranes M3 and M4 are of a magnitude smaller than the commercial high flux F60 dialyzer

69

, it is of significance to note that vitamin B-12 clearance are indeed higher than

commercial AN-69 and Cuprophan membranes 4. Therefore, membranes with higher loading ratio of GO in the nano-composites demonstrate higher clearance of even the middle molecular weight surrogate marker, cytochrome-c. The presence of a greater number of pores (SEM) and the increase of UFR could explain the greater clearance of these solutes.

3.12 Cell Cytocompatibility 3.12.1 MTT Assay On contact with any biomaterials, cells will undergo certain morphological changes in order to stabilize its interface with the material. MTT is the most common and efficient method to determine the cell viability after interaction with the membrane surface. The mitochondrial dehydrogenases of viable cells were usually cleaved selectively to the tetrazolium ring, yielding blue-purple formazan crystals while the level of the reduction of MTT into the colored formazan was measured spectrophotometrically. This reflected the number of cells which were viable. Fig. 15 illustrates the MTT data for the control sample and the membranes incubated in hepatocyte cells for 24 h in a CO2 incubator at 37°C and 5% CO2. The formazan absorbance indicates the quantity of viable cells present on the control (the polystyrene cell-culture plate) as well as those on the prepared membranes. It was observed that, the viability of the cells on all of the modified membranes increased compared with the pristine PEI membranes, which indicated that the 26 ACS Paragon Plus Environment

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addition of the GO nanoparticles might have a positive influence on viability over the period of time and this in turn confirms the cytocompatibility of these MMMs.

3.12.2 Cell Viability Acridine orange /Ethidium orange staining is used to stain the live and dead cells following the incubation of membranes in the cells. The morphological indication of cell apoptosis will infer the viability of cells upon the interaction with the membrane surface. In principle, AO can cleave the intact cell membrane and interact with components of the nucleus and hence only the viable cells will be stained green. The Ethidium bromide is a specific cell stain which imparts the color red to the apoptotic cells due to the impermeable nature of its cell membrane. Fig 15 illustrates the AO/ EtBr staining of hepatocyte cells and from the figures, it is clear that pristine PEI membrane (M0) shows the highest number of apoptotic cells. The increase in the concentration of GO (membranes M3 and M4) in the MMMs decreases the number of apoptotic cells which are visualized by the decrease in red color stained cells. Hence it can be concluded that the number of cells adhered on to the membrane is less when compared to pristine PEI and further adhered cells are viable over the period of time as confirmed by the increase of cells which have imbibed the green stain. Therefore it can be hypothesized that these membranes have a potential to be used not only as hemodialysis membranes but also other bio-artificial organs or organ supports due to their greater cytocompatibility.

4. Conclusion The goal of this study was to prepare novel MMMs by incorporation of hydrophilic GO-PVP nano-composite into the PEI matrix. The presence of these nano-composites on the membrane surface confirmed by FT-IR improved the hydrophilicity of the membrane surface as characterized by lower contact angles and the higher work of adhesion. All of the prepared membranes showed a typical asymmetric structure with lower surface roughness. The integration of the nano-composite leads to an increase in porosity and water flux. This was confirmed by 27 ACS Paragon Plus Environment


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SEM which revealed a greater number longitudinal fully developed finger-like pores. 0.2 wt% GO exhibited higher fouling resistance and greater biocompatibility. These MMMs also had greater biocompatibility with both blood and cells. They exhibited greater protein adsorption resistance, suppressed platelet adhesion, and prolonged clotting time. It can be seen that the uremic toxin removal efficiency increased as the GO loading concentrations increased.

Overall the membrane M3 exhibited a high UFR, high uremic toxin removal efficiency shown increased cell viability, prolonged clotting times, suppressed complement activation, lowered protein adsorption and low platelet activation at the same time maintaining sufficient clearance of the uremic toxins. These show a much lower loss of essential proteins like serum albumin and smoother surface (than M4) which could prevent it from damaging blood cells in an in-vivo study. Hence, it can be confirmed that the introduction of GO-PVP in the membrane matrix as a hydrophilic modifier for the PEI membrane is a safe and efficient method for the development of novel materials for blood purification applications.

Acknowledgements This study was supported by the Instrument Development Program (IDP) of the Department of Science and Technology (DST), Government of India. The authors would like to thank Dr. S. Senthil Kumar for valuable discussions regarding the cell culture studies. We gratefully acknowledge the help extended by Dr. Saravana Babu, Center for Toxicology and Developmental Research (CEFT), SRMC for permitting use of lab space for the cytocompatibility studies. We would also like to thank Mr. Philip George T for his valuable inputs.


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List of Tables 1. Physical characterization of the membranes. 2. Permeation, rejection and fouling parameters of the membranes.

Table 1: Physical characterization of the membranes. Membrane Code

GO (%)

M0 M1 M2 M3 M4

0.00 0.025 0.05 0.1 0.2

Mean Pore Size (10-9 m)

Porosity (%)

32 36 44 52 76

62.8 66.6 68.4 72.5 77.2

Roughness Parameters from AFM (nm) Ra

Rq

20.6(±2.6) 8.4(±1.8) 9.6(±2) 11.8(±1.4) 13.6(±1.6)

Rz

32.6(±1.8) 126.6(±22.4) 14.4(±2.4) 88.4(±14.6) 16.6(±3.2) 92.6(±18.8) 18.2(±2.4) 122.4(±25.5) 21.2(±1.6) 144.8(±12.8)

Table 2: Permeation, rejection and fouling parameters of the membranes. Membrane Code

BSA retention (%)

UFR (mL/h·m2·mmHg)

M0 M1

99 (±0.5) 99 (±0.5)

6.8 8.4

FRR (%) 1 2nd 3rd Run Run Run 76 71 64.5 79 76 75.5 st



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M2 M3 M4

98 (±0.5) 98 (±0.5) 95 (±1)

14.6 26.8 35.0

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84 88 92

81 86 89

78 86 87.5

List of Figures Graphical Abstract 1. 2. 3. 4. 5. 6. 7.

FT-IR and Raman Spectroscopy of pristine Graphite and Graphene Oxide. HR-TEM, XRD and UV Spectroscopy of the prepared Graphene Oxide. Optical Images of the membrane samples. Surface Chemical structures (FT-IR) of the membranes. Cross sectional morphology of the membranes using SEM. AFM imaging of top surface morphology of the membranes. Sessile drop contact angle and work of adhesion for the pristine PEI and modified membranes. 8. BSA and FnG adsorption onto the membranes. 9. Platelet Adhesion by SEM- (A) M0, (B) M1, (C) M2, (D) M3, (E) M4 and (F) LDH Assay results. 10. Thrombin- Antithrombin- III(TAT)generation. 11. Activated partial thromboplastin time (aPTTs) and prothrombin times (PTs) of the membranes. 12. Plasma Recalcification Time (PRT) of the different membranes. 13. Classical pathway and Alternate pathway complement activity of the membrane exposed to the serum. 14. Permeation coefficient of Urea, Vitamin B-12 and Cytochrome-c through the membranes. 15. MTT Assay and Live/Dead cell Staining images.


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Graphical Abstract



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Fig 1: FT-IR and Raman Spectroscopy of pristine Graphite and Graphene Oxide.


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Fig 2 : HR-TEM, XRD and UV Spectroscopy of the prepared Graphene Oxide.



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Fig 3: Optical Images of the membrane samples.


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Fig 4: Surface Chemical structures (FT-IR) of the membranes.



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Fig 5: Cross sectional morphology of the membranes using SEM.


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Fig 6: AFM imaging of top surface morphology of the membranes.



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Fig 7: Sessile drop contact angle and work of adhesion for the pristine PEI and modified membranes.


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Fig 8: BSA and FnG adsorption onto the membranes.



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Fig 9: Platelet Adhesion by SEM- (A) M0, (B) M1, (C) M2, (D) M3, (E) M4 and (F) LDH Assay results.


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Fig 10: Thrombin- Antithrombin- III(TAT)generation.



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Fig 11: Activated partial thromboplastin time (aPTTs) and prothrombin times (PTs) of the membranes.


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Fig 12: Plasma Recalcification Time (PRT) of the different membranes.



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Fig 13: Classical pathway and Alternate pathway complement activity of the membrane exposed to the serum.


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Fig 14: Permeation coefficient of Urea, Vitamin B-12 and Cytochrome-c through the membranes.



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Fig 15: MTT Assay and Live/Dead cell Staining


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Graphene Oxide Nanocomposite Incorporated Poly(ether imide

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