characterising nanoporous carbon adsorbents for … · biological application to chronic kidney...

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Copyright copy 2012 American Scientific PublishersAll rights reservedPrinted in the United States of America

Journal ofBiomaterials and Tissue Engineering

Vol 2 40ndash47 2012

Characterising Nanoporous Carbon Adsorbents forBiological Application to Chronic Kidney Disease

Susan Sandeman1lowast Malin Petersson2 Sara Wiezell2 Carol Howell1 Gary Phillips1Yishan Zheng1 Steve Tennison3 Oleksandr Kozynchenko3 and Sergey Mikhalovsky1

1Biomaterials and Medical Devices Research Group School of Pharmacy and Biomolecular Sciences Huxley BuildingUniversity of Brighton Lewes Road Brighton East Sussex BN2 4GJ UK

2Department of Pharmacology The Sahlgrenska Academy at the University of GothenburgP O Box 431 405 30 Gothenburg Sweden

3MAST Carbon International Guildford Surrey GU3 2AF UK

The study characterises a range of advanced MAST carbons derived from pyrolysed porous phe-nolic resins for use in biological applications considering how the porosity of the carbons impactsboth the methods used to assess potential cytotoxicity and the removal of biological moleculesrelevant to kidney disease The large surface area and pore volume available for adsorption makeboth the microporous and the mesoporous MAST carbons suitable for creatinine removal but notfor removal of the small but highly water soluble urea However only the highly adsorbent MASTcarbons with meso- and macroporosity were able to remove the high molecular weight inflammatorycytokine IL-6 It is the adsorption of the larger biological toxins represented by IL-6 in this studyand not removed by purely microporous carbons or in current haemodialysis therapy which couldmost significantly contribute to the augmentation of current haemoperfusion therapy

Keywords Activated Carbon Adsorbent Chronic Kidney Disease Uraemic Toxins

1 INTRODUCTION

The large adsorptive potential of activated carbons makesthem ideal for the removal of drugs and endogenous tox-ins from biological systems1 Yatzidis first demonstratedthe effective medical use of traditional carbons in anextracorporeal system for the treatment of renal failurein 19642 However complications associated with finesrelease necessitated the coating of carbon with a biocom-patible cellulose membrane34 The coatings provided abarrier that limited the size of biological molecules thatcould be removed and restricted the use of carbon adsor-bents to the treatment of poisoning1

There are a range of medical conditions which areinfluenced by the activity of microbial toxins and theassociated excessive activity of endogenous inflammatorymolecules whose uncontrolled activity is detrimental tohuman health5ndash9 These toxins tend to be middle moleculeslarger than 500 Da in size and cannot be removed by tradi-tional microporous coated carbons alone However newsynthetic MAST carbons with a larger and more definedporosity are capable of removing these larger biologically

lowastAuthor to whom correspondence should be addressed

active molecules by adsorptive processes into meso- andmacroporous domains10 MAST carbons offer a superioradsorptive potential for the treatment of medical conditionsincluding sepsis renal failure liver failure and heart dis-ease since complications associated with these disease pro-cesses often result from the build up and activity of largerbiologically active molecules which are not removed bycurrent filtration based treatment strategiesOne application for adsorptive therapy is in the treat-

ment of chronic kidney failure where new approachesto overcome the deficiencies in haemodialysis arenecessary1112 When renal failure occurs a large number ofcompounds normally excreted by the kidneys remain in thebody These substances known as uremic retention solutesor uremic toxins if they are biologically active are groupedinto small water soluble compounds middle moleculesand protein-bound compounds13 Haemodialysis removessome of the small water soluble toxins including crea-tinine and urea but fails to remove significant amountsof the protein bound and large molecular weight toxinsparticularly14 The patho-physiology of progressive renalfailure is linked to the effect which these poorly removedmolecules have on body systems Creatinine is a smallwater soluble guanidine produced by muscle breakdown

40 J Biomater Tissue Eng 2012 Vol 2 No 1 2157-908320122040008 doi101166jbt20121026

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Sandeman et al Characterising Nanoporous Carbon Adsorbents for Biological Application to Chronic Kidney Disease

and is almost exclusively cleared by the kidneys Urea isthe main product of nitrogenous compound metabolismin the liver and its removal reflects efficient removal ofsmall ionic species such as potassium While other ure-mic retention solutes are emerging as more important indi-cators of negative disease outcome creatinine and ureaclearance rates may be used to assess the extent of kidneydisease and the efficiency of dialysis treatment In addi-tion the inflammatory cytokine IL-6 can be used to assessthe porosity required to remove larger molecular speciesThese include the biologically active molecules that appearto play a prominent role in the pathogenesis of inflamma-tory diseaseThe study describes the characterisation of a range of

synthetic medical grade activated MAST carbon beadsand the way in which the tailored beads can be assessedfor use in biomedical applications considering porositycytotoxicity and the removal of model biological mark-ers relevant to kidney disease The unique porosity of themeso- and macroporous MAST carbons themselves sug-gest they are ideally suited as medical adsorbents Theycombine the superior adsorptive capacity of activated car-bons with the specifically augmented capacity of MASTcarbons for larger biological toxins central to the patho-physiology of disease states associated with dysregulatedinflammatory activation

2 EXPERIMENTAL DETAILS

21 Carbons

Four types of medical grade synthetic carbon bead withvarying porosity were synthesised by MAST Carbon Inter-national (Guildford Surrey UK) from phenolic resin usingpatented technology The development of micropores is amajor contributor to surface area and was controlled byphysical activation burn-off using CO2 Transport poros-ity and the development of mesoporous and macroporousdomain size and volume was controlled by varying thepore former concentration in the composition of pheno-lic resin pre-cursor15 A commercially available carbonmade from peach stone was included as a control (NationalAcademy of Sciences Kiev Ukraine)

22 Physical Characterisation

221 Scanning Electron Microscopy

The macroporous surface structure of the carbons wasvisualised using a Jeol JSM-6310 scanning electron micro-scope (SEM) set at an accelerating voltage of 5 kV The5 carbon samples were mounted on an aluminium stub fit-ted with an adhesive carbon pad and sputter coated withpalladium using a Polaron SC7640 sputter coater

222 Nitrogen and Mercury Porosimetry

The pore size distribution pore volume and surface areaof the carbons was analysed using nitrogen gas adsorp-tion at 77 K and mercury porosimetry on an Autosorb gassorption analyser and an automatic PoreMasterreg mercuryintrusion porosimeter (Quantachrome Instruments) respec-tively The carbon surface area calculations were carriedout using BET theory

23 Cytotoxicity Studies

231 MTS and LDH Assays

Carbon leachate was prepared by incubating heat sterilisedcarbon samples in Dulbeccorsquos Modified Eagles Medium(DMEM) for 24 hours using 10 ml media per gram ofcarbon on a shaking incubator at 22 C A PVC polymercoated with dibutyltin maleate was included as a posi-tive cytotoxic control The extracts were used undiluted ordiluted with an equal volume of media to give leachateconcentrations of 100 50 25 and 0 V79 hamster lungfibroblasts (ECACC no 86041102) were grown in DMEMsupplemented with foetal calf serum (10 vv) and incu-bated at 37 C in a 5 CO2 atmosphere Cells were pas-saged by standard trypsinisation and seeded into 96 welltissue culture plates at a density of 104 cells per well Cellswere incubated for 24 hours at 37 C Media was removedand carbon leachate was added to the wells using serialdilutions of 100 50 25 and 0 for each carbon leachateCells were incubated for a further 24 hours MTS reagent(Promega) was diluted 15 in phenol red free media and100 l was added to each well Plates were incubatedfor 2 hours at 37 C then absorbance was measured at490 nm using a BioTek ELX800 spectrophotometer TheLDH assay was carried out by setting up plates as for theMTS assay Following cell incubation with carbon leachatefor 24 hours cells were lysed and 50 l of lysed cellsuspension was transferred to a second 96 well plate towhich 50 l of LDH substrate solution was added Theplates were incubated in the dark at room temperaturefor 30 minutes 50 l of stop solution was added andabsorbance was measured as for the MTS assay Assayswere carried out in triplicate Results were analysed forstatistical significance using Students t-test

232 LiveDead Staining and Fluorescent Imaging

V79 cells were seeded into the wells of a 24 well plate ata seeding density of 05times104 cells per well and incubatedat 37 C in a 5 CO2 atmosphere for 24 hours Mediawas removed and carbon leachate was added to each wellin triplicate diluted by 50 in media The cells were incu-bated for a further 24 hours Calcein acetoxymethyl ester(calcein-am) was diluted 104 in PBS Media was removedfrom the cells and 500 l calcein-am solution was added

J Biomater Tissue Eng 2 40ndash47 2012 41

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Characterising Nanoporous Carbon Adsorbents for Biological Application to Chronic Kidney Disease Sandeman et al

to each well Plates were incubated for 20 minutes andsolution was removed Cells were rinsed with PBS andviewed by confocal microscopy Calcein-am is cell mem-brane permeable and is hydrolysed to calcein by esterasesin the cytoplasm of living cells Calcein has a stable greenfluorescence which can be monitored using excitation andemission spectra of 494 nm and 520 nm respectively

24 Measuring the Removal of ModelBiological Compounds CreatinineUrea and Cytokine IL-6

241 Creatinine Adsorption

A standard curve was established for creatinine concen-trations of up to 08 mM in Tyrode buffer The kineticsof creatinine adsorption by the carbons was established byincubation of 30 mg of each of the test carbons in 5 mlof 08 mM creatinine solution over time following car-bon equilibration in buffer for 24 hours Samples wereremoved after 5 30 60 and 90 minutes and creatinine con-centration remaining was measured by UV-spectroscopy ata wavelength of 245 nm in order to establish differencesin creatinine removal by the carbons over time The capac-ity of each of the test carbons for creatinine was measuredby carbon incubation with different creatinine concentra-tions 0015 g of each carbon was incubated in 4 ml Tyrodebuffer for 24 hours at 25 C on an orbital incubator Bufferwas removed and each carbon was incubated in 5 ml ofcreatinine solution at concentrations of 05 1 2 4 6 1216 mM for a further 24 hours Carbon 2 3 and 4 werealso incubated in 20 mM and 30 mM creatinine solutionFollowing incubation with the carbon samples creatininesolutions were removed and analysed by UV-spectroscopyat a wavelength of 245 nm The amount of creatinineabsorbed per gram of carbon was calculated according tothe following equation

qe = V Co minusCem

qe is the amount of adsorbate per gram of carbon at equi-librium Co is the initial concentration of creatinine Ce is

NH2H2N

O

OH

NO O

+ H2O 2NH3 + CO2

Urea

NH3+ OClndash + 2

sodium nitroprusside

indophenol

urease

phenol

hypochlorite

ammonia

Fig 1 The reaction of urea catalysed by urease forming ammonia which in turn reacts with phenol and hypochlorite to indophenol in the presenceof sodium nitroprusside

the concentration reached V is the volume of solutionL and m is the amount of carbon added in grams

242 Urea Adsorption

Urea concentration was measured according to a pre-viously established method by the reaction outlinedin Figure 116 The adsorptive capacity of the test carbonsfor urea was measured by incubating 5 different concentra-tions of urea (1 3 5 10 15 mM) made up in Tyrode bufferwith 0025 g of each test carbon for 24 hours at 37 C onan orbital incubating shaker The concentration of urea wasmeasured by mixing 15 l of urea sample with 75 l ofa 10 mg mlminus1 urease solution made up in 40 mM HEPESbuffer 1 ml of phenol nitroprusside solution was addedfollowed by 1 ml of hypochlorite reagent and then 5 ml ofwater The solution was incubated for 30 minutes at 37 CAbsorbance of the solution was measured at a wavelengthof 625 nm using UV-spectrometry All tests were carriedout in triplicate and the amount of urea removed per gramof carbon was calculated as for creatinine

243 IL-6 Adsorption

Mast carbons 1 3 and 4 were used to assess the effectof pore size distribution on the kinetics of cytokine IL-6removal over time 01 g of each carbon was weighedinto eppendorf tubes in triplicate and 1 ml of phosphatebuffered saline was added Carbons were incubated for1 hour on an orbital shaking incubator at 25 C PBSwas removed and 1 ml of human plasma spiked with1000 pg mlminus1 IL-6 was added to each carbon sample Thesamples were incubated at 37 C on an orbital incubatingshaker and 220 l aliquots were removed at time pointsof 5 45 60 and 90 minutes IL-6 concentration remainingin the plasma samples was measured using an IL-6 ELISAkit (BD Biosciences) according to manufacturerrsquos instruc-tions A 1 in 5 dilution of samples was carried out in orderto calculate IL-6 concentrations from the recombinant IL-6standard curve All data was analysed for statistical signif-icance using Students t-test

42 J Biomater Tissue Eng 2 40ndash47 2012

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3 RESULTS

31 SEM

The MAST carbon beads have a uniform spherical shapewith a diameter of 250 to 500 m (Fig 2) The beadshave a compact surface structure and larger surface macro-pores are not visable in contrast to the control carbonThe highly porous internal structure of the MAST beadscan be seen in the higher resolution image of the tran-sected bead In contrast the carbon control made frompeach stone has a much larger granular size and a moreangular non-uniform shape For biological applications theconsistent smooth spherical nature of the MAST carbonbeads is important because it improves biocompatibilityHaemolysis and blood cell activation are less likely

(a)

(c)

(b)

50microm

Fig 2 SEM images of (a) MAST carbon bead 2 times400) (b) MASTcarbon bead 2 times40) (c) control carbon times40)

0

200

400

600

800

1000

1200

1400

0 02 04 06 08 1

Vol

Ads

(cc

g)

PPo

Carbon 1

Carbon 3

Carbon 2

Carbon 4

Fig 3 Nitrogen adsorption isotherms for MAST carbons 1 to 4

32 Porosimetry

The gas nitrogen adsorption isotherm (at 77 K) for MASTcarbon 1 shows a type I Langmuir isotherm (IUPAC clas-sification) indicative of micropore filing (Fig 3) MASTcarbons 2 3 and 4 produced type IV adsorption isothermscharacteristic of mesoporous carbons Table I contains theporosimetry data for synthetic MAST carbons 1 to 4 andthe control carbon 5 The peach stone derived carbon 5 hasthe lowest calculated surface area of 992 m2 gminus1 Its porevolume is 056 g cm3minus1 which is almost a quarter of thepore volume of the highly mesoporous MAST carbon 2Carbon 1 has the lowest pore volume and the highest den-sity of the MAST carbons and is purely microporous withall pores less than 2 nm in diameter Carbon 2 has thehighest pore volume of 21 cm3 gminus1 predominantly in thearea of small macropores and has a large surface areaof 1483 m2 gminus1 Carbon 3 has some smaller mesoporousdomains with an average diameter of 30 nm a large sur-face area of 1465 m2 gminus1 but a lower pore volume of13 cm3 gminus1 Carbon 4 has a lower pore volume and sur-face area than carbon 2 (1236 m2 gminus1 and 161 cm3 gminus1

respectively) but additionally has larger meso-macroporousdomains with an average pore diameter of 120 nm


The MTS assay can be used to measure the potentiallynegative effect of carbon leachate on cell growth sincethe MTS tetrazolium compound is bioreduced by dehydro-genase enzymes in metabolically active cells into a for-mazan product which can be measured colorimetrically


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Table I In the presence of urease urea forms ammonia which in turnreacts with phenol and hypochlorite to form indophenol in the presenceof sodium nitroprusside

Bead Surface Pore Bulk Mean mesoporediameter area SBET volume density diameter

Sample (m) (m2 gminus1) (cm3 gminus1) (g cm3minus1) (nm)

Carbon 1 250ndash500 1204 069 056 MicroporousCarbon 2 250ndash500 1483 211 021 80Carbon 3 250ndash500 1465 130 038 30Carbon 4 250ndash500 1236 161 018 120Carbon 5 minus 992 056 minus minus

at 490 nm using a plate reader A reduction in signalindicates an adverse effect on cell growth Converselythe LDH assay is used to measure cell death in responseto carbon leachate exposure by the enzymatic conver-sion of a tetrazolium salt to a red formazan product bylactase dehydrogenase which is released into the cellmedium following cell lysis An increase in signal indi-cates an increase in cytotoxic effect Cells grown for6 hours in media that was pre-incubated with the test car-bons for 24 hours showed no significant change in nor-mal cell activity compared to those grown in standardmedia alone (Fig 4(a)) However for the MAST car-bons with the greatest adsorbent capacity cell metabolicactivity significantly declined following 24 hour incubationwith 100 carbon pre-conditioned media (Fig 3(b)) Thereduction in cell metabolic activity measured by reducedMTS absorbance was not observed for control carbon 5Following 50 dilution of pre-conditioned media withnormal media cell metabolic activity returned to controllevels suggesting that the carbons remove media nutrients

(a)

(b)

020406080

100120140

1 2 3 4 5 6

Per

cent

of n

orm

alce

ll ac

tivity

Sample

After 6hincubationAfter 24hincubation

020406080

100120140

1 2 3 4 5 6

Per

cent

nor

mal

cell

activ

ity

Sample

10050250

Fig 4 (a) Percent normal cell activity indicated by live cell MTS con-version to a red formazan product following 6 hours and 24 hours incu-bation with 50 carbon pre-conditioned media for carbon samples 1 to 5tin maleate positive control sample 6 (b) Percent normal cell activityof samples incubated with serial dilutions of media extract for 24 hoursfrom 100 down to 0 (n= 3 mean plusmn SEM)

from the media so that cell growth cannot be sustainedin 100 carbon conditioned media for 24 hours Theobserved reduction in cell metabolic activity is not an indi-cator of carbon cytotoxicity but an indicator of the largeadsorptive capacity of the test MAST carbons In contrastthe cytotoxic control media pre-conditioned in tin maleatereduced cell activity to less than 10 even following 50dilution with normal media prior to addition to the cellsFurther cytotoxicity assays were carried out in carbon con-ditioned media diluted by 50 with normal media Cellsgrown for 6 hours and 24 hours in carbon pre-conditionedmedia diluted with 50 normal media showed no changein activity indicating no leaching of cytotoxic compo-nents from the carbons into the media (Fig 4(a)) In con-trast after 6 hours incubation in tin maleate conditionedmedia cell activity had reduced to 60 that of the controlcells and after 24 hours growth was reduced to 10 thatof the controls The LDH assay showed a similar result(results not shown) Fluorescent staining of live cells withcalcein-am at 2 4 6 and 24 hour time points also showedcomparable cell growth for cells grown in media pre-incubated with carbon and those grown in regular media(Fig 5) In contrast the cells grown in tin maleate pre-incubated media showed a progressive reduction in cellnumber over time

34 Creatinine Removal

A linear relationship between absorbance and creatinineconcentration was observed between concentrations of001 mM and 08 mM (r2 = 099) allowing calculationsof unknown concentrations of creatinine to be calculatedwithin this range The adsorption isotherms for carbon 1to 4 were not significantly different from one another andindicated a high removal capacity for creatinine (Fig 6(a))Carbons 1 to 4 removed up to 25 mMol per gram of car-bon and reached saturation capacity at 16 mM creatinineCarbon 1 had a lower capacity for creatinine removal thancarbons 2 to 4 Peach stone derived carbon 5 reached sat-uration at a concentration of 8 mM creatinine and had amuch lower removal capacity of 1 mmole of creatinineper gram of carbon MAST carbon 2 and 4 showed thefastest removal kinetics over 90 minutes incubation of car-bon in 08 mM creatinine solution adsorbing 01 mmolesper gram of carbon (Fig 6(b)) All of the mast carbonsremoved significantly more creatinine than peach stonederived carbon 5

35 Urea Removal

A linear relationship between indophenol absorbance andurea concentration was observed between concentrationsof 02 mM and 40 mM urea (r2 = 099) The adsorptionisotherms for each of the carbons showed limited removalof urea following incubation of the carbons in increasing


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(d) 24h exposure(a) 2h exposure (b) 4h exposure (c) 6h exposure

Fig 5 Confocal fluorescent images of calcein stained live V79 cells grown in 50 media pre-incubated with carbon 3 over time The images arerepresentative of those seen for each of the cells grown in each of the carbon pre-conditioned media samples Only the tin maleate positive controlinduced cell death and no live cells were observed at 24 hours

concentrations of urea (Fig 7) Only at the highest con-centration of 15 mmoles per gram of carbon was any sig-nificant difference seen in removal of urea by carbon 5 andthe other carbons Carbon 5 reached saturation point at aconcentration of 10 mM urea Carbons 1 to 4 did not reachsaturation point at the highest urea concentration used andremoved 015 mmoles per gram of carbon at 15 mM ureaconcentration

36 IL-6 Removal

No removal of IL-6 was observed by carbon 1 (Fig 8)Carbon 4 rapidly removed the majority of IL-6 after only

(a)

(b)

0

05

1

15

2

25

3

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Ads

orbe

d cr

eatin

ine

inm

mol

e gndash1

carb

on

Ads

orbe

d cr

eatin

ine

inm

mol

e gndash1

carb

on

Creatinine concentration (mM)

Carbon 1

Carbon 2

Carbon 3

Carbon 4

Carbon 5

Control

ndash002

0

002

004

006

008

01

012

1 2 3 4 5 6Carbon

5min

30min

60min

90min

Fig 6 (a) Adsorption isotherm for carbons 1 to 5 showing the aver-age amount of creatinine adsorbed in mmole gminus1 carbon against addedconcentration of creatinine (n = 4 mean plusmn SEM) (b) The adsorptionof creatinine in mmole gminus1 carbon for carbons 1 to 5 from a 08 mMcreatinine solution at time points of 5 30 60 and 90 minutes 6 is thecontrol without carbon (n= 5 plusmn SEM)

ndash002

0

002

004

006

008

01

012

014

016

018

0 2 4 6 8 10 12 14 16

Ads

orbe

d ur

ea in

mm

ole

g-1 c

arbo

n

Urea concentration (mM) added

Carbon 1

Carbon 2

Carbon 3

Carbon 4

Carbon 5

Control

Fig 7 Adsorption isotherm showing the average amount of adsorbedurea in mmole gminus1 carbon following incubation with different concentra-tions of urea (n= 3 plusmn SEM)

ndash1000

100200300400500600700

5 min 45 min 90 min 24hrIL-6

rem

oved

(pg

ml)

carbon1

carbon3

carbon4

plasma +

plasma ndash

Fig 8 Removal of IL-6 by carbon 1 carbon 3 and carbon 4 following5 45 90 minutes and 24 hours incubation of IL-6 spiked plasma withcarbon (n= 3 mean plusmn SEM)

5 minutes incubation with IL-6 spiked human plasmaIn contrast adsorption of IL-6 by carbon 3 was muchslower and the concentration of IL-6 was reduced by only100 pg mlminus1 following 90 minutes incubation with spikedhuman plasma

4 DISCUSSION

The ability of activated carbons to remove a wide range ofbiological molecules by adsorption depends on the prop-erties of both the carbon adsorbent and the biologicalmedium Factors such as carbon particle size surface areapore size distribution pore volume surface chemistry andthe competing properties of other salts and proteins present


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in solution will impact both the adsorptive rate and capac-ity for removal In addition the adsorptive power of thecarbons themselves may influence the methods routinelyused to assess the appropriate use of carbons for medicalapplications The study sought to model the way in whichthe properties of advanced MAST carbons can be assessedfor suitable use in medical adsorbent systems taking intoaccount the impact of the adsorptive capacity of the car-bons themselves on the test procedures employed andmaterial properties which must be modified for biologicalsignificance The use of a synthetic polymer based pre-cursor material allows the production of activated carbonswith a homogeneous and reproducible porous structureunlike that observed using natural pre-cursor materialsMAST activated carbons also possess unique porositycompared to other activated carbons These are obtainedusing novel adaptations of phenolic resin technology toproduce high Novacarb based synthetic mesoporous car-bons of medical grade quality1718

The study compared the adsorptive capacity of fouradvanced phenolic resin derived MAST carbons with dif-ferent surface areas pore size distribution and pore vol-ume with that of a traditional carbon derived from peachstone In addition standard cytotoxicity measurementswere adapted in order to take into account the impact ofcarbon adsorptive capacity on the interpretation of resultsobtainedThe potential cytotoxicity of the carbons was measured

using standard MTS and LDH assays adapted from theEuropean international standard procedures for biologicalevaluation of medical devices part 5 (EN ISO 10993-5)The standard assay requires incubation of potential bioma-terials in media for 24 hours prior to exposure of leachateto cells for 24 hours However when the MAST carbonswere used in this way cell growth was not sustained overthe 24 hour time point in comparison to the media onlyand traditional carbon control Dilution of leachate withnormal media by 50 removed this effect in contrast tothe cytotoxic control The results indicated that the car-bons with a large adsorptive capacity were removing themedia nutrients rather than having a cytotoxic leachingeffect causing reduced cell metabolism on exposure timesof greater than 6 hours In order to allow for this effecta number of adaptations to the assay are possible Thecell exposure time could be reduced to 8 hours in orderto reduce the impact of nutrient removal by the carbonsHowever the effect of the cytotoxic positive control wasreduced more substantially than when leachate was dilutedby 50 and cell exposure was maintained at 24 hourssuggesting that dilution and maintained 24 hour exposuretime was a better adaptation of the assay The results ofthe assay were verified using fluorescent staining of livecells at different time points during incubation with carbonpre-conditioned media A qualitative increase in cell num-ber was observed over the 24 hour incubation for each of

the carbons but not for the cytotoxic control The resultsdemonstrate the importance of using a range of comple-mentary assays to allow accurate interpretation of resultssince at first glance the results suggest a cytotoxic leach-ing effect by the MAST carbons on 24 hour cell exposureIn fact the effect is due to the powerful adsorptive capacityof the MAST carbons themselves in confirmation of resultspreviously obtained showing protein and ion adsorptionfrom cell media by MAST carbons19

The removal of creatinine over 90 minutes occurs morerapidly on incubation with carbons 2 and 4 but is sig-nificantly greater for all of the carbons 1 to 4 than forcontrol carbon 5 The combined large surface area andpore volume influence removal characteristics most signif-icantly in this case Creatinine is a small molecular weightaromatic water soluble compound of 11312 Da with anormal plasma concentration of 011 mM Uremic patientlevels can rise to 12 mM and the experimental resultsindicate that MAST carbons with a capacity to remove25 mmoles gminus1 of carbon could remove clinically sig-nificant levels of creatinine in an adsorbent system Dataindicating removal from human plasma is necessary tosubstantiate the results as removal was measured fromTyrode buffer solution Additionally the production of cre-atinine oxidative metabolite creatol by catalytic oxidationof creatinine on the surface of carbon has been reportedin the literature Creatol is rapidly converted to methyl-guanidine a uremic toxin known to accumulate in endstage renal failure2021 Measurement of carbon removal ofcreatinine by adsorption may be augmented by catalyticconversion of creatinine suggesting caution in the interpre-tation of removal data prior to confirmation of additionalbreakdown products in solution Care should also be takenwhen interpreting biological data since catalysis as wellas adsorption may contribute to the apparent observationof removalUrea removal by all of the carbons was poor Urea is a

small molecular weight molecule of 6006 Da but is highlywater soluble and so remains in solution rather than boundto the hydrophobic carbon surface Since clinical levelsreach 40 mM in uremic patients it is unlikely that carbonremoval of urea by adsorptive processes will prove clini-cally usefulIt may be possible to improve urea removal by the intro-

duction of surface acidic groups since urea may then bindto the negatively charged surface Devices such as theREDY sorbent system delt with urea removal using a ure-ase layer to convert urea to ammonia and carbon dioxidefollowed by ammonia adsorption by a cation exchangerzirconium phosphate22

While creatinine and urea are useful markers whenassessing removal strategies relevant to the treatment ofrenal failure they represent only small water solublemolecules which are readily removed by dialysis and arenot considered to be particularly toxic Of more impor-tance are the larger molecular weight and protein bound


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uremic toxins which are poorly removed by current dial-ysis strategies and are known to have a significant impacton cardiovascular damage associated with renal disease23

The current study used the cytokine IL-6 as a repre-sentative large molecular weight inflammatory protein of26 KDa in order to assess the use of MAST carbonsin the removal of these more clinically relevant groupof molecules The results indicate rapid and sustainedremoval of IL-6 by the mesoporous carbon 4 An 85reduction in IL-6 concentration occurred following 5 min-utes incubation of spiked plasma solution with carbon 4In contrast very little removal occurred on incubationwith carbon 3 with only a 30 reduction in IL-6 con-centration over the 90 minute incubation time The purelymicroporous carbon 1 showed no removal of IL-6 overthe time course of the experiment The results indicatethe importance of larger mesoporous and small macrop-orous domains in the removal of larger biologically activemolecules as observed in previous blood perfusion studieswith carbon based adsorbents10

5 CONCLUSIONS

The study characterises a range of polymer pyrolysedadvanced MAST carbons for use in biological applica-tions considering how the porosity of the carbons impactsboth the methods used to assess potential cytotoxicity andthe removal of biological molecules relevant to kidneydisease The large surface area and pore volume avail-able for adsorption make both the microporous and themesoporous MAST carbons suitable for creatinine removalbut not for removal of the small but highly water solu-ble urea However only the highly adsorbent MAST car-bons with meso- and macroporosity were able to removethe larger molecular weight cytokine IL-6 suggesting theirapplication in the further development of adsorbent sys-tems to augment haemodialysis therapy Activated carbonscurrently in use for medical applications do not possessporous domains larger in size than micropores and smallmesopores The study introduces activated carbon beadswith uniquely tailored meso- and macro porous domainscapable of removing larger biological toxins The capac-ity of the beads for poorly removed larger toxins couldrevolutionise the use of adsorptive technology in med-ical haemoperfusion and dramatically reduce associatedcomplications for patients on renal hepatic and cardiacdirected perfusion systems

References and Notes

1 S V Mikhalovsky Emerging technologies in extracorporeal treat-ment Focus on adsorption Perfusion 18 (1S)47 (2003)

2 H Yatzidis A convenient haemoperfusion micro-apparatus overcharcoal for treatment of endogenous and exogenous intoxication

Its use as an effective artificial kidney Proceedings of the EuropeanDialysis and Transplantation Association (1964) Vol 1 p 83

3 D O Cooney (ed) Activated Charcoal in Medical ApplicationsMarcel Deeker New York (1995)

4 J Kellum L Kong M P Fink L A Weissfeld M DonaldD M Yealy M R Pinsky J Fine A Krichevsky R L Deludeand D Angus Understanding the inflammatory cytokine responsein pneumonia and sepsis Arch Intern Med 167 1655 (2007)

5 C Tetta R Bellomo P Inguaggiato M Wratten and C RoncoEndotoxin and cytokine removal in sepsis Ther Apher Dial 6 109(2002)

6 S Hisataka Extracorporeal endotoxin removal for the treatment ofsepsis Ther Apher Dial 7 108 (2003)

7 M Nagaki and H Moriwaki Implication of cytokines Roles oftumor necrosis factor in liver injury Hepatology Research 38 S19(2008)

8 C G Antoniades P A Berry J A Wendon and D Vergani Theimportance of immune dysfunction in determining outcome in acuteliver failure Journal of Hepatology 49 845 (2008)

9 L Li J Pan and Y Yu Development of sorbent therapy for mul-tiple organ dysfunction syndrome (MODS) Biomed Mater 2 R12(2007)

10 S R Sandeman C A Howell S V Mikhalovsky G J PhillipsA W Lloyd J G Davies S R Tennison A P Rawlinson andO P Kozynchenko Inflammatory cytokine removal by an activatedcarbon device in a flowing system Biomaterials 29 1638 (2008)

11 T W Meyer J W T Peattie J D Miller D C Dinh N SRecht J L Walther and T H Hostetter Increasing the clearanceof protein-bound solutes by addition of a sorbent to the dialysateJ Am Soc Nephrol 18 868 (2007)

12 D J Malik G L Warwick M Venturi M Streat K Hellgardtand N Hoenich Preparation of novel mesoporous carbons for theadsorption of an inflammatory cytokine (IL-1beta) Biomaterials25 2933 (2004)

13 R Vanholder European Uraemic Toxin Work Group Review onuremic toxins Classification concentration and interindividual vari-ability Kidney Int 63 1934 (2003)

14 R Vanholder S Van Laecke and G Glorieux What is new in ure-mic toxicity Pediatr Nephrol 23 1211 (2008)

15 O P Kozynchenko V V Strelko A J Blackburn andS R Tennison Porous Carbons US patent 7850942 B2 (2010)

16 A L Chaney and E P Marbach Modified Reagents for Determina-tion of Urea and Ammonia Clin Chem 8 130 (1961)

17 S R Tennison Phenolic-resin-derived activated carbons ApplCatal A General 173 289 (1998)

18 S T Meikle Novel Carbon Based Materials for Use in Extracor-poreal Systems Dissertation University of Brighton Brighton UK(2009)

19 L M Barnes G J Phillips J G Davies A W Lloyd E CheekS R Tennison A P Rawlinson O P Kozynchenko and S VMikhalovsky The cytotoxicity of highly porous medical carbonadsorbents Carbon 47 1887 (2009)

20 E M Smith S Affrossman and J M Courtney The catalytic oxi-dation of creatinine by activated carbon Carbon 17 149 (1977)

21 H Krawczyk Production of uremic toxin methylguanidine from cre-atinine via creatol on activated carbon J Pharm Biomed Anal49 945 (2009)

22 M Roberts The regenerative dialysis (REDY) sorbent systemNephrology 4 275 (1998)

23 P Stenvinkel J J Carrero J Axelsson B LindholmO Heimburger and Z Massy Emerging biomarkers for evaluatingcardiovascular risk in the chronic kidney disease patient How donew pieces fit into the uremic puzzle Clin J Am Soc Nephrol3 505 (2008)

Received 23 May 2011 Accepted 28 July 2011


RESEARCH

ARTIC

LE


and is almost exclusively cleared by the kidneys Urea isthe main product of nitrogenous compound metabolismin the liver and its removal reflects efficient removal ofsmall ionic species such as potassium While other ure-mic retention solutes are emerging as more important indi-cators of negative disease outcome creatinine and ureaclearance rates may be used to assess the extent of kidneydisease and the efficiency of dialysis treatment In addi-tion the inflammatory cytokine IL-6 can be used to assessthe porosity required to remove larger molecular speciesThese include the biologically active molecules that appearto play a prominent role in the pathogenesis of inflamma-tory diseaseThe study describes the characterisation of a range of

synthetic medical grade activated MAST carbon beadsand the way in which the tailored beads can be assessedfor use in biomedical applications considering porositycytotoxicity and the removal of model biological mark-ers relevant to kidney disease The unique porosity of themeso- and macroporous MAST carbons themselves sug-gest they are ideally suited as medical adsorbents Theycombine the superior adsorptive capacity of activated car-bons with the specifically augmented capacity of MASTcarbons for larger biological toxins central to the patho-physiology of disease states associated with dysregulatedinflammatory activation

2 EXPERIMENTAL DETAILS

21 Carbons

Four types of medical grade synthetic carbon bead withvarying porosity were synthesised by MAST Carbon Inter-national (Guildford Surrey UK) from phenolic resin usingpatented technology The development of micropores is amajor contributor to surface area and was controlled byphysical activation burn-off using CO2 Transport poros-ity and the development of mesoporous and macroporousdomain size and volume was controlled by varying thepore former concentration in the composition of pheno-lic resin pre-cursor15 A commercially available carbonmade from peach stone was included as a control (NationalAcademy of Sciences Kiev Ukraine)

22 Physical Characterisation

221 Scanning Electron Microscopy

The macroporous surface structure of the carbons wasvisualised using a Jeol JSM-6310 scanning electron micro-scope (SEM) set at an accelerating voltage of 5 kV The5 carbon samples were mounted on an aluminium stub fit-ted with an adhesive carbon pad and sputter coated withpalladium using a Polaron SC7640 sputter coater

222 Nitrogen and Mercury Porosimetry

The pore size distribution pore volume and surface areaof the carbons was analysed using nitrogen gas adsorp-tion at 77 K and mercury porosimetry on an Autosorb gassorption analyser and an automatic PoreMasterreg mercuryintrusion porosimeter (Quantachrome Instruments) respec-tively The carbon surface area calculations were carriedout using BET theory


231 MTS and LDH Assays

Carbon leachate was prepared by incubating heat sterilisedcarbon samples in Dulbeccorsquos Modified Eagles Medium(DMEM) for 24 hours using 10 ml media per gram ofcarbon on a shaking incubator at 22 C A PVC polymercoated with dibutyltin maleate was included as a posi-tive cytotoxic control The extracts were used undiluted ordiluted with an equal volume of media to give leachateconcentrations of 100 50 25 and 0 V79 hamster lungfibroblasts (ECACC no 86041102) were grown in DMEMsupplemented with foetal calf serum (10 vv) and incu-bated at 37 C in a 5 CO2 atmosphere Cells were pas-saged by standard trypsinisation and seeded into 96 welltissue culture plates at a density of 104 cells per well Cellswere incubated for 24 hours at 37 C Media was removedand carbon leachate was added to the wells using serialdilutions of 100 50 25 and 0 for each carbon leachateCells were incubated for a further 24 hours MTS reagent(Promega) was diluted 15 in phenol red free media and100 l was added to each well Plates were incubatedfor 2 hours at 37 C then absorbance was measured at490 nm using a BioTek ELX800 spectrophotometer TheLDH assay was carried out by setting up plates as for theMTS assay Following cell incubation with carbon leachatefor 24 hours cells were lysed and 50 l of lysed cellsuspension was transferred to a second 96 well plate towhich 50 l of LDH substrate solution was added Theplates were incubated in the dark at room temperaturefor 30 minutes 50 l of stop solution was added andabsorbance was measured as for the MTS assay Assayswere carried out in triplicate Results were analysed forstatistical significance using Students t-test

232 LiveDead Staining and Fluorescent Imaging

V79 cells were seeded into the wells of a 24 well plate ata seeding density of 05times104 cells per well and incubatedat 37 C in a 5 CO2 atmosphere for 24 hours Mediawas removed and carbon leachate was added to each wellin triplicate diluted by 50 in media The cells were incu-bated for a further 24 hours Calcein acetoxymethyl ester(calcein-am) was diluted 104 in PBS Media was removedfrom the cells and 500 l calcein-am solution was added


RESEARCH

ARTIC

LE






qe = V Co minusCem


NH2H2N

O

OH

NO O

+ H2O 2NH3 + CO2

Urea

NH3+ OClndash + 2


indophenol

urease

phenol

hypochlorite

ammonia



242 Urea Adsorption


243 IL-6 Adsorption



RESEARCH

ARTIC

LE


3 RESULTS

31 SEM


(a)

(c)

(b)

50microm


0

200

400

600

800

1000

1200

1400

0 02 04 06 08 1

Vol

Ads

(cc

g)

PPo

Carbon 1

Carbon 3

Carbon 2

Carbon 4


32 Porosimetry






RESEARCH

ARTIC

LE







(a)

(b)

020406080

100120140

1 2 3 4 5 6

Per

cent

of n

orm

alce

ll ac

tivity

Sample


020406080

100120140

1 2 3 4 5 6

Per

cent

nor

mal

cell

activ

ity

Sample

10050250





35 Urea Removal



RESEARCH

ARTIC

LE





36 IL-6 Removal


(a)

(b)

0

05

1

15

2

25

3

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Ads

orbe

d cr

eatin

ine

inm

mol

e gndash1

carb

on

Ads

orbe

d cr

eatin

ine

inm

mol

e gndash1

carb

on


Carbon 1

Carbon 2

Carbon 3

Carbon 4

Carbon 5

Control

ndash002

0

002

004

006

008

01

012

1 2 3 4 5 6Carbon

5min

30min

60min

90min


ndash002

0

002

004

006

008

01

012

014

016

018

0 2 4 6 8 10 12 14 16

Ads

orbe

d ur

ea in

mm

ole

g-1 c

arbo

n


Carbon 1

Carbon 2

Carbon 3

Carbon 4

Carbon 5

Control


ndash1000

100200300400500600700


rem

oved

(pg

ml)

carbon1

carbon3

carbon4

plasma +

plasma ndash



4 DISCUSSION



RESEARCH

ARTIC

LE











RESEARCH

ARTIC

LE




5 CONCLUSIONS





























RESEARCH

ARTIC

LE






qe = V Co minusCem


NH2H2N

O

OH

NO O

+ H2O 2NH3 + CO2

Urea

NH3+ OClndash + 2


indophenol

urease

phenol

hypochlorite

ammonia



242 Urea Adsorption


243 IL-6 Adsorption



RESEARCH

ARTIC

LE


3 RESULTS

31 SEM


(a)

(c)

(b)

50microm


0

200

400

600

800

1000

1200

1400

0 02 04 06 08 1

Vol

Ads

(cc

g)

PPo

Carbon 1

Carbon 3

Carbon 2

Carbon 4


32 Porosimetry






RESEARCH

ARTIC

LE







(a)

(b)

020406080

100120140

1 2 3 4 5 6

Per

cent

of n

orm

alce

ll ac

tivity

Sample


020406080

100120140

1 2 3 4 5 6

Per

cent

nor

mal

cell

activ

ity

Sample

10050250





35 Urea Removal



RESEARCH

ARTIC

LE





36 IL-6 Removal


(a)

(b)

0

05

1

15

2

25

3

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Ads

orbe

d cr

eatin

ine

inm

mol

e gndash1

carb

on

Ads

orbe

d cr

eatin

ine

inm

mol

e gndash1

carb

on


Carbon 1

Carbon 2

Carbon 3

Carbon 4

Carbon 5

Control

ndash002

0

002

004

006

008

01

012

1 2 3 4 5 6Carbon

5min

30min

60min

90min


ndash002

0

002

004

006

008

01

012

014

016

018

0 2 4 6 8 10 12 14 16

Ads

orbe

d ur

ea in

mm

ole

g-1 c

arbo

n


Carbon 1

Carbon 2

Carbon 3

Carbon 4

Carbon 5

Control


ndash1000

100200300400500600700


rem

oved

(pg

ml)

carbon1

carbon3

carbon4

plasma +

plasma ndash



4 DISCUSSION



RESEARCH

ARTIC

LE











RESEARCH

ARTIC

LE




5 CONCLUSIONS





























RESEARCH

ARTIC

LE


3 RESULTS

31 SEM


(a)

(c)

(b)

50microm


0

200

400

600

800

1000

1200

1400

0 02 04 06 08 1

Vol

Ads

(cc

g)

PPo

Carbon 1

Carbon 3

Carbon 2

Carbon 4


32 Porosimetry






RESEARCH

ARTIC

LE







(a)

(b)

020406080

100120140

1 2 3 4 5 6

Per

cent

of n

orm

alce

ll ac

tivity

Sample


020406080

100120140

1 2 3 4 5 6

Per

cent

nor

mal

cell

activ

ity

Sample

10050250





35 Urea Removal



RESEARCH

ARTIC

LE





36 IL-6 Removal


(a)

(b)

0

05

1

15

2

25

3

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Ads

orbe

d cr

eatin

ine

inm

mol

e gndash1

carb

on

Ads

orbe

d cr

eatin

ine

inm

mol

e gndash1

carb

on


Carbon 1

Carbon 2

Carbon 3

Carbon 4

Carbon 5

Control

ndash002

0

002

004

006

008

01

012

1 2 3 4 5 6Carbon

5min

30min

60min

90min


ndash002

0

002

004

006

008

01

012

014

016

018

0 2 4 6 8 10 12 14 16

Ads

orbe

d ur

ea in

mm

ole

g-1 c

arbo

n


Carbon 1

Carbon 2

Carbon 3

Carbon 4

Carbon 5

Control


ndash1000

100200300400500600700


rem

oved

(pg

ml)

carbon1

carbon3

carbon4

plasma +

plasma ndash



4 DISCUSSION



RESEARCH

ARTIC

LE











RESEARCH

ARTIC

LE




5 CONCLUSIONS





























RESEARCH

ARTIC

LE







(a)

(b)

020406080

100120140

1 2 3 4 5 6

Per

cent

of n

orm

alce

ll ac

tivity

Sample


020406080

100120140

1 2 3 4 5 6

Per

cent

nor

mal

cell

activ

ity

Sample

10050250





35 Urea Removal



RESEARCH

ARTIC

LE





36 IL-6 Removal


(a)

(b)

0

05

1

15

2

25

3

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Ads

orbe

d cr

eatin

ine

inm

mol

e gndash1

carb

on

Ads

orbe

d cr

eatin

ine

inm

mol

e gndash1

carb

on


Carbon 1

Carbon 2

Carbon 3

Carbon 4

Carbon 5

Control

ndash002

0

002

004

006

008

01

012

1 2 3 4 5 6Carbon

5min

30min

60min

90min


ndash002

0

002

004

006

008

01

012

014

016

018

0 2 4 6 8 10 12 14 16

Ads

orbe

d ur

ea in

mm

ole

g-1 c

arbo

n


Carbon 1

Carbon 2

Carbon 3

Carbon 4

Carbon 5

Control


ndash1000

100200300400500600700


rem

oved

(pg

ml)

carbon1

carbon3

carbon4

plasma +

plasma ndash



4 DISCUSSION



RESEARCH

ARTIC

LE











RESEARCH

ARTIC

LE




5 CONCLUSIONS





























RESEARCH

ARTIC

LE





36 IL-6 Removal


(a)

(b)

0

05

1

15

2

25

3

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Ads

orbe

d cr

eatin

ine

inm

mol

e gndash1

carb

on

Ads

orbe

d cr

eatin

ine

inm

mol

e gndash1

carb

on


Carbon 1

Carbon 2

Carbon 3

Carbon 4

Carbon 5

Control

ndash002

0

002

004

006

008

01

012

1 2 3 4 5 6Carbon

5min

30min

60min

90min


ndash002

0

002

004

006

008

01

012

014

016

018

0 2 4 6 8 10 12 14 16

Ads

orbe

d ur

ea in

mm

ole

g-1 c

arbo

n


Carbon 1

Carbon 2

Carbon 3

Carbon 4

Carbon 5

Control


ndash1000

100200300400500600700


rem

oved

(pg

ml)

carbon1

carbon3

carbon4

plasma +

plasma ndash



4 DISCUSSION



RESEARCH

ARTIC

LE











RESEARCH

ARTIC

LE




5 CONCLUSIONS





























RESEARCH

ARTIC

LE











RESEARCH

ARTIC

LE




5 CONCLUSIONS





























RESEARCH

ARTIC

LE




5 CONCLUSIONS





























characterising nanoporous carbon adsorbents for … · biological application to chronic kidney...

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