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Emerging technologies in extracorporeal treatment: focus on adsorption Sergey V Mikhalovsky Perfusion 2003; 18; 47 DOI: 10.1191/0267659103pf627oa The online version of this article can be found at: http://prf.sagepub.com/cgi/content/abstract/18/1_suppl/47

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Perfusion 2003; 18: 47 ¡/54

Emerging technologies in extracorporeal treatment: focus on adsorption Sergey V Mikhalovsky University of Brighton, Brighton, UK As an extracorporeal technique for blood purification, haemoadsorption was introduced in the early 1960s along with other physico-chemical methods. The problem of poor biocompatibility of uncoated adsorbents was resolved by coating adsorbent granules with haemocompatible membranes. Use of coated adsorbents instead of uncoated ones reduces the efficiency of haemoperfusion.

As a result, for many years the use of adsorption was limited to only acute poisoning. Since the 1990s interest in the use of adsorbents in extracorporeal medical devices has been rising again. In this paper some recent developments in synthesis and application of novel uncoated medical adsorbents are discussed. Perfusion (2003) 18, 47 ¡/54.

Haemoadsorption, or haemoperfusion (HP) as a novel extracorporeal technique, was introduced by Yatzidis in the early 1960s.1,2 Although the initial results were very successful, this procedure induced hypotension, hypocalcaemia, hypokalaemia, hypoglycaemia and thrombocytopenia.3 The most severe potential complication was the release of fine particles from the carbon granules, causing microemboli.4 Coating of adsorbent granules with haemocompatible membranes was suggested as a way to solve these problems.5,6 The goal of improving adsorbent biocompatibility was thus achieved, but at the cost of the adsorbent performance. Adsorption of high-molecular weight (HMW) solutes is particularly affected. A 3 ¡/5-m m thick membrane virtually cuts off HMW molecules and significantly reduces adsorption of ‘middle molecules’, with molecular mass between 300 and 15 000 Dalton. 7 Removal of ‘middle molecules’ is essential, as they play an important role in the development of many pathological conditions. Commercial HP columns contain charcoal produced from peat or pitch and coated with cellulose (Adsorba 300 C and Adsorba 150C; Gambro, Swedwn), poly-HEMA (Hemosorba; Asahi Medical, Tokyo, Japan and Nextron Medical Technologies, Fairfield, NJ, USA) or heparin hydrogel (Clark R&D, USA).8 ¡ 10 Use of coated adsorbents instead of uncoated adsorbents dramatically reduces the efficiency of HP, both in terms of adsorption capacity and rate of

adsorption. As a result, HP has been limited in use to only acute poisoning with certain low-molecular toxins. 11 As many small molecules are proteinbound in the blood, they cannot cross the membrane coating; hence, HP over coated adsorbents would be efficient in removing only protein-free solutes of low molecular mass.

Address for correspondence: Sergey Mikhalovsky, School of Pharmacy and Biomolecular Sciences, University of Brighton, Cockcroft Building, Lewes Road, Brighton, BN2 4GJ, UK. E-mail: [email protected]

# Arnold 2003

Alternative extracorporeal methods based on dialysis and filtration have proved to be more versatile than HP over coated adsorbents, and took over; although in the 1960s and 1970s all three groups of methods seemed to have similar opportunities for clinical use.12 Since the 1990s, however, interest in the use of adsorbents in extracorporeal medical devices has been rising again for at least three reasons: i) inefficiency of other methods in the treatment of some autoimmune diseases, severe sepsis and multiorgan failure.13 ii) Shift of the priorities in the desirable outcome of treatment towards providing good quality of life for patients who require regular treatment, such as patients with chronic renal failure. Use of dialysis dramatically increased life expectancy of these patients, but the quality of life remains unsatisfactory.14 iii) The extracorporeal methods based on dialysis and filtration are expensive; they have already become a significant economic burden on healthcare services, which is projected to increase further due to the problem of ageing population in the developed world.15 In this paper some recent developments in syntheses and applications of medical adsorbents are discussed.


10.1191/0267659103pf627oa

New technologies in extracorporeal treatment SV Mikhalovsky

48 Table 1 Extracorporeal techniques ¡/ mechanisms and efciency of blood cleansing Removal of:

Haemodialysis

Haemofiltration

Haemoperfusion

Fluid/water Solute Small molecules ‘Middle molecules’ Large molecules

Ultrafiltration Diffusion High Low Low to none

Ultrafiltration Convection Moderate to high Variable Low to none

None Adsorption Variable Potentially high Variable

Adsorption versus other physicochemical methods in extracorporeal techniques Adsorption as a method of blood cleansing has a series of advantages over other physicochemical methods (Table 1). Provided they have sufficiently large pores, uncoated adsorbents can remove solutes of any molecular size. Unlike dialysis and filtration, not to mention drug-based therapy, adsorption potentially can remove toxic substances without introducing anything else instead.16 If no fluid removal from the body is necessary, adsorption is more cost effective than dialysis or filtration, as a significant volume of expensive replacement solutions is required in the latter. It also appears that, in many cases, dialysis or filtration removes middle and high molecular solutes, such as inflammatory cytokines and endotoxin via adsorption and retention on the filter or hollow-fibre surface rather than by convection or diffusion through the membrane.17,18

Adsorbents and mechanisms of adsorption Adsorbents retain the adsorbed molecules by chemical or physical attraction forces. Physical forces include all types of Van der Waals and hydrophobic interactions, whereas chemical interaction involves some sort of chemical bond formation between the surface and the adsorbed species.19 Adsorption or surface retention of solutes takes place in a network of pores. According to the International Union of Pure Applied Chemistry (IUPAC) classification, pores are divided into three groups according to their size of entry: micropores (less than 2 nm); mesopores (2 ¡/50 nm); and macropores (over 50 nm).20 This classification is not entirely mechanistic and it reflects different mechanisms of adsorption and retention in pores of different sizes.21 Industrial applications of activated carbon as an adsorbent mostly exploit its micropores, hence, most carbons have well-developed microporous structure, but few mesopores. For medical applications, mesopores also become relevant as toxins of protein origin; ‘middle molecules’ and low molecu-

lar protein-bound substances can not be adsorbed in micropores due to the size mismatch. Use of coated haemoadsorbents, however, makes mesopores almost inaccessible. Although chemical interaction is more specific than physical, it is seldom used to achieve higher selectivity of adsorption in medical applications because, in complex biological media, there will always be a number of molecules of different origins competing for the chemical adsorption site against the target molecules. Better results are achieved when a molecule with a specific affinity (bioligand) with the target molecule is immobilized on the surface; for example, either partner of the antibody ¡/antigen pair.22 High affinity is realised via a combination of electrostatic and hydrophobic interactions plus steric complementarity of the two molecules rather than covalent chemical bond formation. By their chemical composition, medical adsorbents can be divided into three major groups: i) activated carbon (AC); ii) synthetic and natural organic polymers, such as polystyrene and cellulose; and iii) inorganic adsorbents, such as silica and oxides of titanium and zirconium.23 Activated, or active carbon is the most powerful adsorbent among all the materials, as it has the largest surface area ¡/ in excess of 2000 m2 /g ¡/ and pore volume ¡/ up to 1.8 cm3 /g.21 The name ‘activated carbon’ is a generic term referring to a very wide range of materials that significantly differ by the shape and size of their granules or fibres, pore structure, surface chemistry, mechanical properties, chemical composition and the nature of the material-precursor. This fact seems to have been ignored by clinicians with respect to medical applications, as the ACs originally tested in HP have never been designed specifically for this purpose. It should not be surprising, therefore, that so many complications were observed during HP over uncoated carbons. It is rather surprising that the first HPs went so smoothly, because the adsorbents used in these treatments were technical grade granular activated carbons taken ‘off-the-shelf’. Pretreatment of these carbons was limited to washing with hydrochloric acid and deionized water and priming with saline.1,24 Although AC as such is a



49

non-specific physical adsorbent, it demonstrates strong p ¡/p interactions with cyclic organic molecules, which make it a more selective adsorbent towards aromatic amino acids, phenols, aspirin and other substances containing aromatic rings, compared with other adsorbents.25 The surface chemistry of AC is very complex and a variety of polar or ionogenic functional groups have been found on its surface. The overall mechanism of adsorption on AC comprises a multitude of ion exchange, hydrogen bonding, formation of surface complexes, molecular adsorption and hydrophobic interactions. Synthetic organic polymers tested in extracorporeal methods are mainly based on cross-linked polystyrene with or without surface functional groups.26 Nonfunctionalized polymers are nonpolar and preferentially adsorb hydrophobic organic solutes, whereas functionalized polymers act as cation or anion exchangers and preferentially adsorb ionized molecules and ions. The main use of the polysaccharides agarose and cellulose is as matrices for biospecific and immunoadsorbents.23 The ability of inorganic adsorbents to adsorb various ions is most frequently exploited in their medical applications. A multiadsorbent system, REDY (RenalSolutions, IN, USA), introduced in the 1970s, used zirconium phosphate, zirconia and alumina in addition to AC.27 A special case is the use of silica as a matrix for the immobilization of Protein A to produce an immunoadsorbent for removing immunoglobulin (Ig) G and immune complexes, which recently obtained Food and Drug Administration (FDA) approval for treatment of rheumatoid arthritis.28,29 Biocompatibility of inorganic matrices is generally poor compared with

other adsorbents and they are not used for direct contact with blood.

Use of adsorbents in combined extracorporeal systems In commercial HP columns only coated adsorbents are used (Table 2).30 ¡ 36 In order to reduce the volume of replacement fluid and ultimately operational costs, adsorbent regeneration and recycling of ultrafiltrate or dialysate have been used in clinical experiments since the 1970s.37,38 As adsorbent particles do not come in direct contact with blood or plasma, the issue of their biocompatibility is much less strict and no coating is usually needed. Some of the devices utilizing adsorptive regeneration of ultrafiltrate/dialysate have already been commercialized or have passed the pilot stage (Table 2). In fact, there is one particular extracorporeal technique that can not possibly work without adsorptive purification of biological fluids, which is a (bio)artificial liver device.39 At present, without a charcoal adsorption column, which removes lowmolecular hepatic toxins, such a device is inefficient. In the molecular adsorbent recycling system (MARS; Teraklin, Germany), human serum albumin is used for removing toxins from blood in the primary circuit; being a liquid sorbent, it has to be regenerated in the secondary circuit using solid adsorbent particles (Table 2). Columns with biospecific uncoated adsorbents are used in plasmapheresis for treatment of some immune diseases and familial hypercholesterolae-

Table 2 Non-specic HP and adsorbent-assisted extracorporeal systems System

Manufacturer/developer

Adsorbent

Coating

BioLogic-DT, BioLogic DTPF, Liver Dialysis Unit Molecular adsorbent recycling system (MARS) BetaSorb

Hemocleanse, HemoTherapies, USA

Charcoal»/cation exchanger

Uncoated [30]

Teraklin, Germany

Uncoated [31]

Plasorba BR-350 Hemosorba CH-350 (HP)

Asahi Medical Co Ltd, Japan Asahi Medical Co Ltd, Japan; Nextron Medical Technologies, USA RenalSolutions, USA

Human serum albumin, ion exchange resin and AC Hydrated cross-linked polystyrene resin Anion-exchange resin Charcoal

Uncoated [23, 27]

Gambro, Sweden

Charcoal, inorganic ion exchangers Charcoal

Coated

[8]

Clark R&D, Inc., USA

Charcoal

Coated

[10]

Bellco SpA, Italy

Charcoal»/neutral polystyrene resin Neutral resin, activated carbon, cellulose anion exchanger Charcoal

Uncoated [34]

Sorbent Hemodialysis System (SHD), REDY system Adsorba 150C, Adsorba 300C (HP) Clark Biocompatible Hemoperfusion System (HP) Selecta Microspheres based detoxification systems (MDS) YTS (HP)

RenalTech International, USA

University of Krems, Austria Aier, China


Coated

Reference

[32]

Uncoated [33] Coated [9]

Uncoated [35] Coated

[36]


50

mia, but these columns are rather expensive and the number of treated patients is small.28,29,40

Uncoated AC for direct HP Although adsorption techniques are returning to the medical practice, further progress requires developing uncoated adsorbents with high haemo/biocompatibility to allow direct contact with blood. In addition to its superior adsorption features, AC has a series of other advantages over other adsorbents in this respect. Firstly, it is a rigid material that does not swell in water or other solvents, unlike polymers, and does not require special pretreatment in such a solvent. It is also easier to maintain stable flow characteristics of a biological fluid through a column packed with carbon granules than through a column with soft polymer granules. Second, AC is chemically inert compared with polymers, as it does not contain any plasticizer, catalyst or monomer that can leak from the material into the bloodstream. The chemical inertness of carbon is a direct consequence of the physical conditions in which it is synthesized. Physical activation, or development of the pore structure, occurs by treating the carbonized material at 800 ¡/10008C with carbon dioxide or steam.21 Under these conditions no organic matter can exist, being converted either into carbon or gaseous products. (A common and persistent belief that carbon is carcinogenic has its origin in the fact that some volatile products of incomplete combustion of coal are carcinogenic, but this process has nothing to do with the production of AC)41 As much as a few hundred grams of AC could be consumed orally by a person who suffers from acute poisoning without any negative consequences, confirming the very low toxicity and chemical inertness of this substance.42 Pyrolytic carbon is used in artificial organs, such as mechanical heart valves, showing excellent biocompatibility.43,44 Chemically, pyrolytic and activated carbons are the same substance, making it reasonable to expect good biocompatibility of AC. Chemical inertness may not be a key issue in comparing AC with polymers as haemoadsorbents in the treatment of acute poisoning, when only a few HP procedures suffice. It becomes a serious problem for chronic patients requiring regular treatment for years, such as chronic renal patients on long-term dialysis. The success of the pure carbon heart valve can be attributed to the quality of the material, which perhaps was the first medical grade carbon specifically designed for such applications. Similarly, AC of medical grade can be produced from synthetic

Figure 1 Pore size distribution in (a) commercial AC used in Adsorba 300C HP column and (b) AC produced from cross-linked polystyrene. Pore size distribution is estimated from low-temperature adsorption of nitrogen using Gemini II adsorption analyser (Micromeritics). Surface area of AC in (a) is 900 m2 /g; pore volume¾/0.51 mL/g; surface area of AC in (b) is 1200 m2 /g; pore volume¾/1.15 mL/g.

polymers, which are made totally free from uncontrolled impurities. Moreover, carbon produced by polymer pyrolysis retains the porous structure of its precursor, which allows for formation of a unique mesoporous structure in the AC in addition to common micropores (Figure 1).45,46 The presence of mesopores makes this material an efficient adsorbent for middle and large molecules (Table 3). Table 3 Adsorption of TNFa and lipopolysaccharide (LPS) from model solution, % of initial concentration AC

TNFa*

LPS**

From polystyrene From polyvinylpyridine

96 82

89 40

* From 500 pg/mL solution in Tyrode’s buffer with 0.1% bovine serum albumin, after 360 minutes; 0.3 adsorbent per 15 mL solution. Concentration of TNFa was measured with ELISA kit (IDS Ltd). ** from 10 m g/mL solution of LPS from E. coli serotype 0.111: B4 (Sigma-Aldrich) containing 10% v/v foetal calf serum, after 360 minutes; 0.3 adsorbent per 15 mL solution. Concentration of LPS was measured by the response of macrophages to LPS using nitrite and protein assays.



51

Figure 2 Scanning electron micrographs of (a) coated commercial AC used in Adsorba 300C HP column and (b) uncoated AC produced from cross-linked polystyrene. Note that the coated surface of AC in (a) is smooth, whereas the exposed carbon surface in the place where the coating is damaged is rough. In contrast, the surface of the uncoated polymer pyrolysed AC in (b) is smooth. Downloaded from http://prf.sagepub.com at PENNSYLVANIA STATE UNIV on April 10, 2008 © 2003 SAGE Publications. All rights reserved. Not for commercial use or unauthorized distribution.


52 Table 4 K » and Na » in plasma and erythrocytes of the blood of patients with tuberculosis before and after direct HP over uncoated polymer pyrolysed AC (courtesy of Prof AN Burov); HP column with 500 mL of AC, blood ow 150 mL/min, 90 min Concentration (mmol/L)

Before DHP

After DHP

AC from polyvinylpyridine K » plasma K » erythrocytes Na » plasma Na » erythrocytes

4.49/0.4 78.89/3.6 1329/5.0 17.79/2.4

4.09/0.2 76.09/2.3 1279/5.2 17.29/2.2

AC from polystyrene K » plasma K » erythrocytes Na » plasma Na » erythrocytes

4.59/0.1 78.29/3.0 1309/3.5 15.39/1.4

4.09/0.3* 72.09/2.9* 1229/3.4* 17.29/1.3

* Significant difference (P B/0.05)

Precursor polymers are synthesized by emulsion polymerization as uniform spherical granules and this shape and surface smoothness are retained by AC (Figure 2). Due to the high mechanical strength of carbon beads, they do not generate microparticles, which eliminates the threat of microembolism. A simple pretreatment of AC by washing it with a solution of the same mineral composition as blood, such as Tyrode’s buffer, blocks ionogenic functional groups that may be on the carbon surface, preventing or significantly reducing the loss of inorganic ions (Table 4) reported in the early papers on HP.3 No significant changes in biochemical parameters of blood after direct HP over uncoated polymer pyrolysed activated carbon have been reported.16

Future prospects adsorbents?

¡

well. Although this is a legitimate point, so far no problems or side effects due to the loss of nutrients or other useful substances after HP have been reported, nor has such a loss been significant. If adsorbents are to be used regularly for the treatment of chronic patients, this may become a problem and improving selectivity of adsorption is required. Selectivity of adsorption can be increased using a single or a combination of several approaches, such as regulation of pore size, chemical modification of the surface hydrophilicity/hydrophobicity, introduction of ionogenic groups and surface charge alteration. In principle, adsorbents can be ‘tailor made’ for specific interactions with the toxic solute. The most selective adsorbents can be synthesized by the immobilization of a bioligand with a high affinity towards target molecules. Use of uncoated AC for direct HP opens an interesting opportunity to design biospecific adsorbents based on carbon matrix. Despite its chemical inertness, it is possible to introduce reactive functional groups onto the carbon surface and subsequently attach bioligands, but the efficiency of this approach needs further research.47 In order to use selective adsorption, it is necessary to know what substances have to be removed, and this is not always possible. So nonspecific adsorbents will continue to play their role in the treatment of diseases with unknown aetiology.23

Acknowledgements /

towards biospecific

One of the most common criticisms of the use of extracorporeal methods, particularly adsorption, is the lack of selectivity in their action, i.e., in addition to the target substances removed from the bloodstream, many useful components can be removed as

The author thanks Dr OP Kozynchenko, MAST Carbon Ltd (Surrey, UK) for characterization of carbon samples; Dr AN Burov (Institute of Tuberculosis, Moscow, Russia) for data presented in Table 4, and also his colleagues Prof AW Lloyd, Dr JG Davies, Dr GJ Phillips and Ms MC Murphy from the University of Brighton (Brighton, UK) for the discussion of this paper.

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