Graphene-Based Materials for the Fast Removal of Cytokines from

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Graphene-Based Materials for the Fast Removal of Cytokines from Blood Plasma Mykola Seredych, Bernard Haines, Viktoriia Sokolova, Paul Cheung, Fayan Meng, Lon Stone, Lyuba Mikhalovska, Sergey Mikhalovsky, Vadym N. Mochalin, and Yury Gogotsi ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00151 • Publication Date (Web): 18 Jul 2018 Downloaded from http://pubs.acs.org on July 21, 2018

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ACS Applied Bio Materials

Graphene-Based Materials for the Fast Removal of Cytokines from Blood Plasma

Mykola Seredych,1 Bernard Haines,1 Viktoriia Sokolova,1 Paul Cheung,1 Fayan Meng,1 Lon Stone,2 Lyuba Mikhalovska,3 Sergey Mikhalovsky,3 Vadym N. Mochalin,4, 5 and Yury Gogotsi1*

1

Department of Materials Science & Engineering and A.J. Drexel Nanomaterials Institute,

Drexel University, Philadelphia, Pennsylvania 19104, United States 2

Consultants Rx, Dana Point, California 92629, United States

3

School of Pharmacy and Biomolecular Sciences, University of Brighton, Lewes Road, Brighton,

BN2 4GJ, United Kingdom 4

Department of Chemistry, Missouri University of Science & Technology, Rolla, Missouri

65409, United States 5

Department of Materials Science & Engineering, Missouri University of Science & Technology,

Rolla, Missouri 65409, United States

*

Corresponding author. E-mail address: [email protected] (Y. Gogotsi) 1

ACS Paragon Plus Environment

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ABSTRACT There is a range of medical conditions, which include acute organ failure, bacterial and viral infection and sepsis, that result in over-activation of the inflammatory response of the organism and release of pro-inflammatory cytokines into the bloodstream. A fast removal of these cytokines from blood circulation could offer a potentially efficient treatment of such conditions. This study aims at the development and assessment of novel biocompatible graphene-based adsorbents for blood purification from pro-inflammatory cytokines. These graphene-based materials were chosen on the basis of their surface accessibility for small molecules further facilitated by the interlayer porosity, which is comparable to the size of the cytokine molecules to be adsorbed. Our preliminary results show that graphene nanoplatelets (GnP) exhibit high adsorption capacity, but they cannot be used in direct contact with blood due to the risk of small carbon particles release into the bloodstream. Granulation of GnP using poly(tetrafluoroethylene) (PTFE) as a binder eliminated an undesirable nanoparticle release without affecting the GnP surface accessibility for the cytokines molecules. The efficiency of pro-inflammatory cytokine removal was shown using a specially designed flow-through system. So far, GnP proved to be among the fastest acting and most efficient sorbents for cytokine removal identified to date, outperforming porous activated carbons and porous polymers.

KEYWORDS: graphene nanoplatelets, human blood plasma, cytokines, sepsis, adsorption

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INTRODUCTION Cytokines play an important role in pathogenesis of some very serious life-threatening conditions such as sepsis, multi-organ failure and acute kidney injury.1 Usually triggered by bacterial infection, these conditions are characterized by an over-expression of cytokines, both proinflammatory and anti-inflammatory mediators of immune response.2-6 Sepsis is associated with a massive and rapid increase in blood levels of specific cytokines – mediators of inflammatory response such as ‘pro-inflammatory’ interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α).7 Cytokines are proteinaceous molecules that regulate the inflammatory response and, as the disease aggravates, cause a “cytokine storm” known as hypercytokinemia.8-12 High mortality of patients has been associated with high levels of both pro- and anti-inflammatory cytokines.13 Due to the need of urgent treatment, a broad-spectrum antibiotic therapy is currently the predominant treatment of sepsis in hospitals. However, antibiotics are not efficient against viral infections, nor they mediate the inflammatory response. Severe side effects and complications may lead to an up to 9% increase in mortality rate per each hour of improper treatment.14,15 To present, no conclusive treatment of sepsis and its complications has been established.16 Reducing the cytokine level, in particular that of proinflammatory cytokines IL-6, IL-8, and TNF-α, has been considered as a potential treatment of such conditions.7 It has been reported that sepsis treatment with antibodies against TNF-α resulted in a modest improvement in the mortality rate,17 but many other results are inconclusive and, moreover, the use of selective methods targeting individual cytokines is expensive and unlikely to address the complex issue of regulating the immune response in sepsis. Non-selective 3



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methods of extracorporeal blood purification used in renal replacement therapy have been suggested for a rapid removal of cytokines from blood. Among them, dialysis and ultrafiltration and their combinations have been used most frequently.18,19 In comparison with these methods, another extracorporeal technique, adsorbent-based blood or blood plasma purification known as hemoperfusion (HP) or plasma perfusion respectively, has been seldom used. However, a thorough analysis of the mechanisms of cytokine removal by dialysis or filtration revealed that the main route of their elimination from the bloodstream is via adsorption by the surface of dialysis hollow fibers or filter membranes respectively.20 It revived interest in the cytokine removal by adsorption in a hemoperfusion column.21 In order to treat sepsis in the intensive care unit (ICU), fast adsorption kinetics and high cytokine adsorption capacity are key to patient survival. Therefore, methods designed to reduce the concentrations of various cytokines simultaneously prove to be more effective.22-24 Several adsorbents have been designed to remove cytokines (typically IL-1 family, IL-6, IL-8, IL-10, and TNF-α) in extracorporeal devices.7, 25-42 Special interest has been focused on removal of TNF-α as it is strongly correlated with patient survival,25 but this pro-inflammatory cytokine also is the most difficult to adsorb due to its large size, as it exists as a trimer in blood circulation (9.4 nm × 9.4 nm × 11.7 nm). Filtration can be done using hemodialysis or hemofiltration, an extracorporeal treatment which involves the diffusion or convection of solutes across a hollow fiber or semipermeable membrane respectively.26 Nevertheless, these methods often have complications, including excessively rapid fluid removal through ultrafiltration, which can lead to low blood pressure and exposure of the circulatory system to microbes.26,27 Another limitation is a rapid decline of the molecular 4


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weight cut-off of membranes during treatment due to the narrowing and partial blockage of the pores because of blood plasma protein adsorption. Hemoperfusion, on the other hand, does not pose such problems: (i) adsorbents have a much larger specific surface area and porosity than membranes and hollow fibers, which is unlikely to be saturated or pores blocked during the HP session, and (ii) the fluid loss is minimal and much smaller volume of replacement fluid is required.26-28 In hemoperfusion, to reduce blood clotting in the adsorbent column and address the biocompatibility issues of the adsorbent material, the blood plasma can be separated from the whole blood and perfused through the adsorbent column and then reintroduced to the whole blood. In principle, HP is a less costly and more efficient detoxifying option than hemodialysis or hemofiltration.29 A number of adsorbents were assessed in vitro for cytokine removal. They include polymer-derived carbons (PDCs),30,38,39 carbide-derived carbons (CDCs),31,32,36 polymer adsorbents (polystyrene and cellulose),21,33-35,41,42 or modified inorganic materials37 due to their high specific surface area and large mesopore volume. CDCs, synthesized from silicon carbonitride, with 254 m2/g specific surface area of pores larger than 5.0 nm, showed high removal efficiency for cytokines from the blood plasma.36 It was shown that the mesopores with the diameter between 5.0 nm and 9.4 nm are critical thresholds for accommodation of the adsorbed biomolecules, such as IL-6 and TNF-α, with the removal efficiency 80% and 60%, respectively.36 The efficiency of TNF-α, IL-6 and IL-8 adsorption by activated carbons obtained from a mesoporous phenol-formaldehyde-aniline resin was compared with commercially available adsorbents, such as Adsorba® 300C (cellulose-coated activated carbon Norit RBX),30 5



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Cytosorb® (polystyrene-divinylbenzene porous beads coated with polyvinylpyrrolidone),31 and CXV (activated carbon adsorbent from CECA).35 The carbons with larger mesopore size (~40 nm) showed a greater uptake of TNF-α. The adsorption of TNF-α, IL-6, and IL-8 by PDCs showed that the carbon with the greatest degree of activation (60% burn-off) can remove 99100% of cytokines present in concentrations up to 5,000 pg/mL in blood plasma of a septic patient.30 However, removal of inflammatory cytokines using carbon monoliths showed that the TNF-α removal efficiency dropped from 90% to 0% over the 90-min flow cycle at a flow rate of 500 µL/min.38 Authors linked it to saturation of the larger mesopores by the adsorbed TNF-α, while significant removal of other cytokines (IL-6, IL-1β and IL-8) was still observed. A similar PDC with very large specific surface area (3460 m2/g) and total specific pore volume (2.32 cm3/g) performed exceptionally well having an adsorption capacity of 36.2 and 94.8 ng/g for TNF-α and IL-6, respectively.39 Table S1 summarizes information on various materials used for cytokines removal. Recently, we demonstrated the biocompatibility of vacuum annealed graphene nanoplatelets and observed low cytotoxicity and rapid removal of pro-inflammatory cytokines in batch experiments.43 The structure of this material is fundamentally different from porous carbons, because it has a large external surface accessible to solutes resulting in a much faster adsorption rate. In general, graphene nanoplatelets are among the most efficient sorbents identified to date, greatly outperforming porous activated carbons and porous polymers.21,3039,41,42

Expanded graphite is another low cost carbon material, which can be produced on a large

scale and deserves attention as a potential sorbent.44 6


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The goal of this work is to study the adsorption of cytokines from human blood plasma under dynamic conditions using two-dimensional (2D) carbons such as thermally expanded graphite and two types of graphene nanoplatelets (GnP). It was found that two-dimensional (2D) carbons can remove cytokines much faster and with higher efficiency than highly porous activated carbons, offering a potential to shorten the procedure and to increase the efficiency of blood purification by adsorption.

EXPERIMENTAL SECTION Materials. Three graphene-based materials, expanded graphite (EGr), non-granulated graphene nanoplatelets (GnP), and graphene nanoplatelets granulated by poly(tetrafluoroethylene) binder (GnP:PTFE) were studied for cytokine adsorption. Expanded graphite was obtained from graphite (Superior Graphite, Grade 2900G8) intercalated with H2SO4 (18.4 M) : HNO3 (15.8 M) at 5:1 (v/v) ratio followed by a thermal shock according to the protocol described in Ref.45 First, graphite (50 g) was mixed with concentrated H2SO4 (80 mL) and the mixture was stirred for 10 min. Then, concentrated HNO3 (16 mL) was added to the mixture and it was stirred for additional 2 hours. Afterwards, graphite intercalated with the acids was washed with water to remove any free acids. The obtained graphite intercalation compound (residual graphite bisulfate) was filtered, dried at 120 oC and expanded for 1 min in the furnace preheated to 900 o

C. The volume of the EGr after thermal shock was 98 cm3 and the weight 6.6 g. The expanded

graphite obtained was extensively washed with distilled water until neutral pH. The graphene nanoplatelet material (GnP-300, from xGnPTM, XG Sciences © MI, USA; referred further to as GnP) has been produced by delamination of EGr using sonication.46 GnP 7



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particles are stacks of graphene layers of 1-5 nm in thickness and 1-2 µm in lateral dimensions. Since the lateral dimensions of graphene nanoplatelets are very small, they require granulation in order to improve the column flow rate and prevent any leakage of carbon particulates. In the granulation procedure, PTFE was added as a binder to granulate graphene nanoplatelets dispersed in methanol at 9:1 (w/w) ratio. The obtained mixture was sonicated for 2 h and further stirred continuously for 24 h in order to evaporate methanol. Finally, the slurry was freeze-dried for 24 h to prevent stacking of the graphene nanoparticles. Porosity. Specific surface area and porosity of the adsorbents were analyzed by nitrogen adsorption at -196 oC using a Quadrasorb gas sorption analyzer (Quantachrome Instruments, Boynton Beach, USA). The sample was out-gassed at 100 mTorr and 120 °C for 12 h prior to analysis. The specific surface area, SBET, was calculated using the Brunauer-Emmett-Teller method in the relative pressure range, p/po, from 0.05 to 0.30.47 The total pore volume, Vt, was calculated from the last point of the nitrogen adsorption isotherm at p/po equal to 0.99. Adsorption of cytokines from blood plasma in batch experiments. Fresh frozen human blood plasma (StemCell Technologies, NJ, USA) was defrosted at 37 oC and spiked with the recombinant human cytokines IL-6, IL-8 and TNF-α (BD Biosciences) at a concentration of 400 ± 35 pg/mL, 400 ± 25 pg/mL and 800 ± 50 pg/mL, respectively. Expanded graphite and two graphene nanoplatelet materials, GnP and GnP:PTFE, were used as adsorbents. The adsorbents were weighted (10, 25 or 50 mg) into non-stick microcentrifuge vials and equilibrated in phosphate-buffered saline (PBS, 1.0 mL) by shaking at 150 rpm at room temperature for 24 h. After equilibration in PBS solution, the latter was removed by centrifugation at 14,000 rpm and 8


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1.0 mL of cytokine spiked human plasma was added. The cytokine adsorption time was 60 min. Cytokine-spiked plasma without adsorbents and un-spiked plasma with adsorbents were used as controls. All samples were incubated at 37 oC while shaking at 200 rpm. Then the samples were centrifuged for 5 min at 14,000 rpm and the supernatants were collected and stored at -20 oC prior to measuring the cytokine concentrations by ELISA (BD Biosciences). Supernatants were diluted at ratios 1:4 (IL-6 and TNF-α) and 1:5 (IL-8) in assay diluents prior to analysis. The experiments were conducted three times independently in duplicates. The cytokine concentration was determined from the calibration curve (Figure S1). The optical absorbance was read using Tecan NanoQuant microplate reader (Infinite® M200). The detailed description of the ELISA method is given in the Supporting Information. Adsorption of cytokines from blood plasma under dynamic conditions. Following the batch experiments, granulated GnP:PTFE was chosen as the most promising adsorbent to study the removal of cytokines from blood plasma under dynamic conditions using Masterflex L/S Digital System with Easy – Load II Pump heads. The filtration column (1 mL, non-fluorous polypropylene, Sigma-Aldrich, 57608-U) was packed with GnP:PTFE adsorbent (~270 mg) with the particle size 1-2 mm obtained by sieving and a membrane (polyethylene frit, 20 µm porosity) at the bottom, to ensure good hydrodynamic characteristics of the column. Granules of similar size are used in commercial hemoperfusion columns. Prior to cytokine adsorption, PBS was circulated through the packed filtration column for 2 hours to ensure the uniform environment inside the whole system. Afterwards, the human blood plasma (20 mL) spiked with 400 ± 35 pg/mL, 400 ± 25 pg/mL and 800 ± 50 pg/mL of each cytokine IL-6, IL-8, and TNF-α, 9



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respectively, was passed through the filtration column at a flow rate of 4.5 mL/min. This rate was chosen to provide quick adsorption while avoiding liquid jet induced erosion of the material inside the column. Every 2 mL of the filtrate were collected in separate tubes and stored frozen at -20 oC until used for ELISA analysis. The control measurements of spiked plasma circulated in the loop without adsorbent were taken after each 5 mL of the filtrate passage to ensure there were no changes in the initial concentration. RESULTS AND DISCUSSION Schematic in Figure 1a shows human blood plasma running through a filtration column. The column is packed with the granulated graphene nanoplatelets that adsorb target proteins onto their surface and in between their layers. Granulated graphene nanoplatelets (GnP:PTFE) have a specific surface area of 256 m2/g, which is significantly higher compared to expanded graphite (31 m2/g) (Table 1). These parameters are important to consider because the adsorption efficiency for cytokines IL-6, IL-8, and TNF-α is directly related to the surface area and to the volume of pores that can accommodate these molecules.30,36 GnP:PTFE contains mesopores in the range of 2-12 nm, while EGr has a meso/macroporous structure with the average pore size of 9.8 nm. It is interesting to mention that the porous structure is preserved for granulated GnP:PTFE as compared to the GnP alone. The volume of mesopores for GnP:PTFE is 0.419 cm3/g compared to 0.076 cm3/g for EGr (Table 1). Considering the dimensions of the cytokine molecules: 5 nm × 5 nm ×12.2 nm (IL-6),48 4 nm × 4 nm × 9 nm (IL-8),49 and 9.4 nm × 9.4 nm × 11.7 nm (TNF-α)50 and the dimensions of the pores needed for the adsorption of each cytokine, the pores of adsorbent with the size > 5 nm (IL-6), > 4 nm (IL-8) and > 9.4 (TNF-α) are the most 10


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suitable. The nitrogen adsorption/desorption isotherms of GnP alone and granulated GnP are presented in Figure 1b. It is worth mentioning that for 2D materials, the pore size distribution does not necessarily show the true cumulative pore volume, especially for the materials with large/open pores. Both GnP and GnP:PTFE demonstrate Type II isotherms according to the Brunauer classification.51 The volume of N2 adsorbed significantly increases with the relative pressure suggesting the presence of large mesopores. The mesopores are probably formed between the stacks of graphene layers (1-5 nm in thickness and 1-2 µm in lateral dimensions). Granulation of GnP caused no significant changes in the N2 uptake in the mesoporous range and both graphene materials have remarkable mesopore volume. As expected, the granulated material GnP:PTFE has a slightly (~12%) lower porosity in the range of micro/mesopores than the starting GnP. Despite its lower porosity, GnP:PTFE is more favorable as a candidate material for HP under dynamic adsorption conditions because granulation of the adsorbent improved its flow dynamic quality, which is important to prevent blockage of column, and on the other hand granulation with bioinert PTFE prevents any small carbon particles release into the blood stream without altering its biocompatibility. The morphology of GnP determined from TEM images showed that individual GnP particles are formed of stacks of graphene layers,43 while expanded graphite forms worm-like graphite grains with large size of graphite flakes.52 GnP particles form sub-micron size agglomerates. In addition to porosity and morphology, surface chemistry might play an important role in the specific removal of target cytokines. The XPS results reported in our recently published 11



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paper52 indicated that the surface of GnP is enriched mainly with phenolic/alkoxy/ether groups and the content of oxygen in graphene nanoplatelets is much higher (7.9 at.%) than in the expanded graphite (2.8 at.%). Figure 2 compares the efficiency of cytokines IL-6 (a), IL-8 (b) and TNF-α (c) removal from spiked blood plasma in batch experiments after 60-min incubation using different mass of graphene-based adsorbents (10 mg, 25 mg and 50 mg). This time was determined to be sufficient to reach equilibrium based on the kinetics of bovine serum albumin (BSA) adsorption52 (Figure S3 and Table S3). The adsorption kinetics of cytokines (IL-6, IL-8 and TNF-α) for GnP and GnP:PTFE shows the same equilibrium time as for BSA.43 The data obtained show that both types of graphene nanoplatelets possess very high removal efficiency for IL-8 and IL-6 cytokines. As a result, 10 mg of each adsorbent, GnP or GnP:PTFE adsorbed nearly 100% of IL8 from the spiked plasma. The removal of IL-6 by GnP was also approaching 100% from the spiked plasma, whereas granulated GnP:PTFE demonstrated a lower efficiency (70%) (Figures 2a and 2b). Adsorption of TNF-α by the same mass of GnP and GnP:PTFE adsorbents was much lower and reached only ~20% and ~4% of the initial 800 pg cytokine amount (Figure 2c). To improve the removal efficiency of graphene nanoplatelet adsorbents, a higher mass (25 mg and 50 mg) of both adsorbents was used. Consequently, increasing the amount of GnP from 10 mg to 50 mg raised the removal efficiency of TNF-α from 20% to 98% from the initial 800 pg spiked plasma. The adsorption capacity of the GnP:PTFE adsorbent towards TNF-α also increased significantly, from 4% of the spiked plasma cytokine by 10 mg to ~60% by 50 mg of adsorbent (Figure 2c). Likewise, the removal efficiency of IL-6 by 50 mg of GnP:PTFE increased from 12


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~70% to ~100% (Figure 2a). The expanded graphite (EGr) adsorption of all three cytokines was also studied using 50 mg of adsorbent. The data obtained show that this adsorbent possesses a low capacity, capable of removing less than 27% for all examined cytokines (Figures 2a-c). To improve the removal efficiency of the adsorbents, two approaches could be used: increasing the incubation time or increasing the adsorbent-to-the-volume of the solution ratio. In this work larger amounts of adsorbents were used. Increasing the amount of GnP to 50 mg allowed to us remove nearly all TNF-α from the spiked plasma. In case of GnP:PTFE increasing the amount of adsorbent up to 50 mg helped us to remove nearly all spiked IL-6 during the same incubation time. Removal of TNF-α also increased up to 60% from the spiked amount by 50 mg of the adsorbent. To compare the adsorption capacity of expanded graphite and two types of graphene nanoplatelets, the adsorbed amounts of cytokines were calculated per mg of adsorbent (Figure 3) after 60 min of incubation when the equilibrium was reached. The amount of cytokine per mass of adsorbent was calculated from concentration measured by ELISA in spiked plasma for each cytokine ~415, ~425 and ~800 pg/mL for IL-6, IL-8 and TNF-α, respectively. At the adsorbent mass of 50 mg (Figure 3a), the adsorption capacity of GnP and GnP:PTFE is much higher than that of EGr. Since GnPs demonstrated high adsorption even when used in 10 mg quantities, further experiments were performed with lower masses of the adsorbents to determine their maximum adsorption capacity. At 25 mg and 10 mg, the highest adsorption capacity was measured for GnP towards IL-6 and TNF-α, followed by granulated GnP:PTFE (38.2 ± 2.4 pg/mgads and 32.0 ± 2.0 pg/mgads for IL-6; 19.4 ± 2.0 pg/mgads and 8.8 ± 2.0 pg/mgads for TNF-α, 13



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respectively). However, the highest adsorption capacity for IL-8 was measured in GnP:PTFE followed by GnP (36.1 ± 2.4 pg/mgads and 32.7 ± 1.4 pg/mgads, respectively). The data show that GnP outperformed GnP:PTFE in adsorption of the larger TNF-α with more than double amount of the cytokine adsorbed. For the granulated graphene nanoplatelets at the mass of adsorbent of 50 mg, 25 mg and 10 mg, only IL-8 was completely removed but not IL-6 and TNF-α. Therefore, the data obtained in the batch experiments suggest that the minimal required mass of adsorbent to remove all three cytokines from 1 mL of spiked blood plasma from the lethal to the healthy patient’s levels should be 50 mg under equilibrium. TNF-α has poorly established safe levels, which are strongly dependent on an individual patient. To present, no definitive link between the level of TNF-α in the blood and the septic patient survival has been established and it varies significantly among patients. However, it has been generally accepted that reducing the average rather than peak level of inflammatory cytokines in blood favors the patient survival. Taking 600 pg/mL as the potentially lethal level of TNF-α and 36 – 46 pg/mL as a safe level,53,54 anything in between represents the grey zone where the survival prognosis depends on an individual. Based on the above and considering a 154 lb (70 kg) patient whose blood volume is 5,600 mL (8% body mass, human blood composed of 55% blood plasma) the volume of patient’s blood plasma is 3,080 mL. Based on the adsorption capacity of GnP:PTFE for TNF-α (9 pg/mg), the reduction of the cytokine level in patient’s blood plasma from 600 pg/mL to 40 pg/mL requires about 190 g of the adsorbent. Taking into account the bulk density of GnP:PTFE, which is 0.45 g/cm3, it means that the HP column should have a volume of about 420 mL, which is within the range of commercial HP column volumes of 350-500 mL. 14


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The adsorption results show direct correlation between the amount of the adsorbed cytokines and both the specific surface area and the mesopore volume of adsorbents (Figure 4). The steric effect is another important factor, which plays an important role in cytokine adsorption; the pore size should be sufficiently large to accommodate the adsorbate molecules. If the adsorption capacity for the three cytokines studied is compared at low mass of the adsorbent, 10 mg, the amounts of cytokines adsorbed by GnP from 1 mL of blood plasma are 380 pg, 400 pg and 160 pg of IL-6, IL-8 and TNF-α, respectively. Lower adsorption capacity of GnP:PTFE vs GnP is related to the fact that the same amount of GnP:PTFE has a lower content of GnP than pure GnP, thus resulting in lower specific surface area and pore volume of the former. In addition, some of the spaces between nanoplatelets initially available to TNF-α may be blocked by PTFE. However, the above calculation of the adsorbent capacity towards cytokines was based on the data obtained in batch experiments. Taking into account that hemoperfusion is carried out in the recycling regime, a further study was performed under dynamic conditions using 1 mL column containing ~270 mg of GnP:PTFE. The flow system for this study was designed and built as shown in Figure 5. Figure 6 depicts hemoperfusion and via an adsorbent column and presents the data collected for the removal of cytokines under dynamic conditions. GnP:PTFE showed high removal efficiency (~80%) for IL-6 and IL-8 without reaching saturation or breakthrough point for 12 mL of spiked blood plasma passed through the column. The level of cytokine removal remained at ~80% throughout the adsorption process. However, the breakthrough point for TNF-⍺ was reached at 4 mL and the removal efficiency decreased down 15



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to ~10 % at 12 mL of blood plasma filtered. Despite the significant decrease in TNF-⍺ removal efficiency, the data indicates that saturation has not been reached yet. The large variance in error bars is attributed to the changes in the packing density of the filtration column since the adsorbent particles packed vary in size from 1 to 2 mm.

CONCLUSIONS In this work, we tested expanded graphite (EGr), graphene nanoplatelets (GnP), and granulated graphene nanoplatelets (GnP:PTFE) as adsorbent for the removal of pro-inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α), which are relevant for the development of sepsis. GnP and granulated GnP:PTFE were found to be the most efficient in cytokine adsorption because of their open graphene structure and the interlayer porosity in the range of 2-12 nm, which is similar to the dimensions of the target cytokines. The adsorption capacity of GnP towards IL-6 and IL-8 (from initial ~400 pg/mL spiked plasma) at the ratio of 10 mg of adsorbent per 1 mL of plasma was about 40 pg/mg (nearly 100%). The adsorbent GnP:PTFE showed similar adsorption capacity to IL-8 and a bit lower (~70%) to IL-6 at the same ratio of adsorbent to plasma. Adsorption capacity of both adsorbents towards TNF-α was lower and efficient removal of this cytokine was possible only by increasing the ratio of the amount of adsorbent to the plasma volume (50 mg of adsorbent per 1 mL of spiked plasma), although the initial amount of spiked TNF-α was twice higher than that of IL-6 and IL-8. The limitations of the efficient removal were most likely due to saturation of available platelets’ 16


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surface that can accommodate TNF-α during the adsorption process. Expanded graphite showed a minor decrease in cytokine concentrations in spiked human plasma. The results presented in this study and Table S1 showed that graphene nanoplatelet adsorbents outperform expanded graphite and other carbon-based materials in adsorption rate and capacity towards the cytokines studied. Remarkably, under dynamic conditions, GnP:PTFE proved to be a much more efficient adsorbent for all three cytokines. It was found that the removal efficiency of 80% for IL-6 and IL-8 was maintained for the 12 mL of spiked blood plasma, while TNF-⍺ showed a steady decrease in percentage removed throughout the filtration process (starting at ~80% and decreasing down to ~10%). The results obtained suggest that on a larger scale, 70 g of our adsorbent may treat about 3 L of human blood plasma under dynamic conditions which is an average volume of plasma in a patient. This study has shown that PTFE-bound GnPs have a great potential for the removal of pro-inflammatory cytokines from blood plasma under clinically relevant flow conditions. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: xxx. The supporting material includes materials studied for cytokine removal from spiked human blood plasma, Enzyme-Linked Immunosorbent Assay (ELISA) protocol, and kinetics of BSA adsorption. Notes The authors declare no competing financial interests. 17



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ACKNOWLEDGMENTS This work was supported by the NSF CBET Div of Chem, Bioeng, Env, & Transp Sys Award # 1518999 and British Council and the UK Department for Business, Innovation and Skills through the Global Innovation Initiative.

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CAPTION TO THE TABLES Table 1. Parameters of the adsorbents porous structure calculated from the nitrogen adsorption isotherms (SBET - surface area; Vt - total pore volume; Vmeso - volume of mesopores; Vmic volume of micropores). CAPTIONS TO THE FIGURES Figure 1. a) Schematic showing removal of cytokines with the filtration column packed with granulated GnP:PTFE and illustrating their surface chemistry; b) N2 adsorption/desorption isotherms for the neat graphene nanoplatelets and granulated GnP:PTFE with molecular structures of the target cytokines on the right. Figure 2. Concentrations of IL-6 (a), IL-8 (b) and TNF-α (c) remaining in spiked blood plasma after 60 min of incubation with the different mass of graphene-based adsorbents. Batch tests were conducted three times independently in duplicates. Figure 3. Adsorbed amount of IL-6, IL-8 and TNF-α per milligram of adsorbent under equilibrium conditions (60 min of incubation) with the mass of adsorbent 50 mg (a), 25 mg (b) and 10 mg (c). Batch tests were conducted three times independently in duplicates. 25



Page 26 of 32

Figure 4. Amount of cytokines adsorbed per gram of adsorbent as a function of specific surface area (a) and mesopore volume (b). The mass of adsorbent is 50 mg, spiked with the recombinant human cytokines IL-6, IL-8 and TNF-α at a concentration of 400 ± 35 pg/mL, 400 ± 25 pg/mL and 800 ± 50 pg/mL, respectively and volume 1 mL. Figure 5. a) The in-house built flow system for removal of cytokines from spiked human blood plasma using Masterflex L/S Digital System with Easy – Load II Pump heads. One peristaltic pump with two pump heads was used: blood plasma leaves the patient through the first pump head and returns back to the patient (Flow 1) and then blood plasma circulates in the second pump head (Flow 2) and passes through the filtration column; b) filtration column is packed with adsorbent and c) granulated graphene nanoplatelets (GnP:PTFE) adsorbent. Figure 6. a) Schematic showing removal of cytokines from blood plasma in vivo and b) removal efficiency of IL-6, IL-8 and TNF-⍺ cytokines from spiked blood plasma under dynamic conditions (1 mL filtration column; 4.5 mL/min flow rate) for graphene nanoplatelets granulated with polytetrafluoroethylene (GnP:PTFE). Batch tests were conducted three times independently in duplicates.

26


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Table 1. Parameters of the adsorbents porous structure calculated from the nitrogen adsorption isotherms (SBET - surface area; Vt - total pore volume; Vmeso - volume of mesopores; Vmic volume of micropores). SBET (m2/g)

Vt (cm3/g)

Vmeso (cm3/g)

Vmic (cm3/g)

Average pore diameter (nm)

GnP

286

0.484

0.408

0.076

6.8

GnP:PTFE

256

0.419

0.355

0.064

6.6

EGr

31

0.076

0.076

N/D

9.8

Sample

N/D - Not determined

27



Page 28 of 32

Figure 1. a) Schematic showing removal of cytokines with the filtration column packed with granulated GnP:PTFE and illustrating their surface chemistry; b) N2 adsorption/desorption isotherms for the neat graphene nanoplatelets and granulated GnP:PTFE with molecular structures of the target cytokines on the right.

Figure 2. Concentrations of IL-6 (a), IL-8 (b) and TNF-α (c) remaining in spiked blood plasma after 60 min of incubation with the different mass of graphene-based adsorbents. Batch tests were conducted three times independently in duplicates.

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Figure 3. Adsorbed amount of IL-6, IL-8 and TNF-α per milligram of adsorbent under equilibrium conditions (60 min of incubation) with the mass of adsorbent 50 mg (a), 25 mg (b) and 10 mg (c). Batch tests were conducted three times independently in duplicates.

Figure 4. Amount of cytokines adsorbed per gram of adsorbent as a function of specific surface area (a) and mesopore volume (b). The mass of adsorbent is 50 mg, spiked with the recombinant human cytokines IL-6, IL-8 and TNF-α at a concentration of 400 ± 35 pg/mL, 400 ± 25 pg/mL and 800 ± 50 pg/mL, respectively and volume 1 mL.

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Figure 5. a) The in-house built flow system for removal of cytokines from spiked human blood plasma using Masterflex L/S Digital System with Easy – Load II Pump heads. One peristaltic pump with two pump heads was used: blood plasma leaves the patient through the first pump head and returns back to the patient (Flow 1) and then blood plasma circulates in the second pump head (Flow 2) and passes through the filtration column; b) filtration column is packed with adsorbent and c) granulated graphene nanoplatelets (GnP:PTFE) adsorbent.

30


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Figure 6. a) Schematic showing removal of cytokines from blood plasma in vivo and b) removal efficiency of IL-6, IL-8 and TNF-⍺ cytokines from spiked blood plasma under dynamic conditions (1 mL filtration column; 4.5 mL/min flow rate) for graphene nanoplatelets granulated with polytetrafluoroethylene (GnP:PTFE). Batch tests were conducted three times independently in duplicates.

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Graphical Abstract

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Graphene-Based Materials for the Fast Removal of Cytokines from

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