HSA immobilized novel polymeric matrix as an alternative sorbent in hemoperfusion columns for bilirubin removal

Reactive and Functional Polymers 96 (2015) 25–31

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HSA immobilized novel polymeric matrix as an alternative sorbent in hemoperfusion columns for bilirubin removal Mahdi Kavoshchian a, Recep Üzek a,⁎, Sadık Ahmet Uyanık b, Serap Şenel a, Adil Denizli a a b

Hacettepe Universty, Faculty of Science, Department of Chemistry, Ankara, Turkey 29 Mayıs Satate Hospital, Ankara, Turkey

a r t i c l e

i n f o

Article history: Received 18 May 2015 Received in revised form 31 August 2015 Accepted 11 September 2015 Available online 15 September 2015 Key words: Bilirubin HSA CNBr activation P[HEMA] cryogel

a b s t r a c t HSA immobilized poly(2-hydroxyethylmethacrylate), HSA-P[HEMA], cryogel monolith was examined as an alternative sorbent to be used in hemoperfusion columns for bilirubin removal from serum. The cryogel monolith synthesis was performed by free radical polymerization using MBAA as crosslinker, APS and TEMED as redox pair. Cyanogen bromide (CNBr) was used as a matrix-activating agent for the preparation of immobilized cryogels. Control cryogel monolith, P[HEMA], and HSA-P[HEMA] were characterized by swelling test, SEM images, porosity and surface area measurements, and blood compatibility tests. Activation and immobilization processes were optimized. The removal of bilirubin from plasma samples overloaded with bilirubin was performed using P[HEMA] cryogel monoliths containing different amounts of immobilized HSA in continuous mode. Several factors affecting adsorption capacity of the matrix such as incubation time, HSA concentration, bilirubin concentration in plasma and temperature were analysed. The maximum bilirubin removal from plasma was 25.4 mg/g at 37.5 °C. The desorption agent was 0.1 M NaOH and 1.0 M NaCl containing solution. The reusability was tested for 10 consecutive adsorption–desorption cycles. The adsorption isotherm models and kinetics of process were also studied. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The main bile pigment bilirubin (MW 585 Da) is a linear tetrapyrol, formed by heme degradation [1]. It is transported to liver by albumin, and transformed into a soluble form with glucuronic acid. The production of bilirubin is 250–300 mg/day for adults. Free (unconjugated) bilirubin is highly toxic for several cell types, and physiological processes. The toxicity is due to the inhibition of membrane bound enzymes and damage to membrane integrity in phospholipid membranes [2]. On the other hand, it is also an antioxidant and antiinflammatory, and has preventive effect for cardiovascular, immunologic, and cerebral systems [3]. The normal bilirubin level in serum is 0–0.3 mg/dL for conjugated bilirubin, and 0.3–1.9 mg/dL for total bilirubin. In case of hyperbilirubinemia, the extra bilirubin has the tendency to accumulate and to bond in several tissues including brain causing to kernicterus, thalssemia, Gilbert syndrome, Crigler–Najjar syndrome, encephalopathy, and even to death [4,5]. Four bilirubin fractions were isolated from serum: unconjugated bilirubin, monoconjugated and diconjugated bilirubins, and irreversibly bound bilirubin to protein [6]. Human serum albumin (HSA) is the most abundant protein in blood serum, the typical blood concentration being 5 mg/dL. HSA

⁎ Corresponding author. E-mail address: [email protected] (R. Üzek).

http://dx.doi.org/10.1016/j.reactfunctpolym.2015.09.004 1381-5148/© 2015 Elsevier B.V. All rights reserved.

has two high-affinity binding sites for small heterocyclic or aromatic compounds, 2–3 for long-chain fatty acids, and 2 for metals. The binding sites for bilirubin are involved in IIA and IIIA subdomains [7]. The interaction is between pyrrole rings of bilirubin and aromatic aminoacids of protein due to critical salt bridges between carboxyl groups of bilirubin and lysine and arginine units of protein [8]. The removal of toxic substances from human plasma is targeted in extracorporeal affinity therapy, eliminating the disadvantages of conventional methods such as plasma exchange, phototherapy, hemoperfusion etc. Using affinity carriers as column stationary phase in hemoperfusion systems introduces several advantages such as an increase in performance of procedure, elimination of infection risk, negligible changes in plasma components, co-use of method with drug therapy, and return of patient's blood to his body following the procedure. The cryogels, imprinted cryogels [9,10], particle embedded forms [11,12], modified with biospesific [13,14] or pseudo-biospesific ligands [15,16] are attractive sorbents as packing materials in hemoperfusion columns due to macroporosity, elasticity, and related flow characteristics. Affinity chromatography is often used for purification of biomolecules due to its excellent specificity, ease of operation, yield and throughput [17]. Several affinity adsorbents were tried for bilirubin removal [18–20]. Congo red or Cibacron Blue F3-GA attached P[EGDMAHEMA] microparticles [21,22]. Cibacron Blue F3 GA bound P[GMA] microspheres both in magnetic and nonmagnetic forms [23], Cibacron Blue F3 GA attached polyamide hollow fiber [24] gave capacity values

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ranging between 11.7 mg/g and 241.5 mg/g. In case of HSA-bound P[HEMA] particles in magnetically stabilized flow bed column system 88.3 mg/g capacity was achieved [25]. Albumin immobilized magnetic p(HEMA) particles [26], a cryogel copolymer (HEMA and Nmethacryloyl-L-triptophan methyl ester containing) [27] and bilirubin imprinted, N-methacryloyl-L-tyrosin methyl ester containing particles embedded into p(HEMA) cryogel [28] were the adsorbents worth to be mentioned. The bilirubin adsorption capacity from human plasma was 76.2 mg/g when microporous p(tetrafluoroethylene) capillary, covered with PVA was used [29]. In this study, an affinity adsorbent, HSA immobilized P[HEMA] cryogel monolith was prepared, characterized, and examined for bilirubin removal from plasma. HEMA was chosen as the basic component for synthesis due to its mechanical strength, chemical and biological stability, biocompatibility and inertness. The high affinity between HSA and bilirubin, and the advantages of cryogel format for column studies were combined in synthesis and modification. 2. Materials and methods 2.1. Materials 2-hydroxyethyl methacrylate (HEMA) and N,N,N′,N′-tetramethylethylene diamine (TEMED) were purchased from Fluka (Buchs, Switzerland). N,N′-methylene–bis(acrylamide) (MBAA), ammonium persulfate (APS), human serum albumin (HSA), and cyanogen bromide (CNBr) were obtained from Sigma (St Louis, USA). The other reagents were in analytical grade, and were from Merck (Darmstadt, Germany). Water used in the experiments was purified using a Barnstead (Dubuque, IA) ROpure LP® reverse osmosis unit with a high flow cellulose acetate membrane (Barnstead D2731), followed by a Barnstead D 3804 NANO pure organic/colloid removal and ion exchange packed bed system, giving a conductivity of 18 M Ω/cm. 2.2. Preparation of P[HEMA] cryogel monolith The cryogel was synthesized by redox cryopolymerization method. The monomer phase contained 1.3 mL HEMA, 0.283 g MBAA (crosslinker), and 13.7 mL of water.The monomer solution was degassed under vacuum for about 5 min to remove soluble oxygen and was kept at 0 °C for 20 min. The free radical polymerization was initiated by 0.02 g APS (initiator) and 25 μL TEMED (activator) as redox pair. The 3.0 mL mixture was immediately poured into glass column (5.0 mL, ID 1.0 cm) and frozen at − 16 °C for 24 h. The resulting matrix was thawed at room temperature. The sample was cleaned with deionized water to remove unreacted monomers, and stored in deionized water at 4 °C until use. 2.3. Characterization of PHEMA cryogel monolith The cryogel monolith was characterized by swelling test, BET analysis, porosity measurement, SEM analysis, and blood compatibility tests. 2.3.1. Swelling test: The swelling test was performed using a known mass piece of cryogel incubating in 50.0 mL of deionized water at 25.0 °C in a constant temperature bath for 2 h (previously determined equilibrium swelling time). Then, the sample was dried to constant weight in an oven at 60 °C, and weighed again. The swelling ratio (%) was determined using the following equation: Swelling rationð%Þ ¼ ½ðms −m0 Þ=m0   100 where ms and m0 denoted masses of swollen and dried samples, respectively.

Macropore content of cryogel sample was calculated by determining the masses of swollen and mechanically squeezed samples. Macropore cuntentð%Þ ¼




 mswollen −msqueezed =mswollen  100%

2.3.2. Surface morphology The sample was fixed in 2.5% glutaraldehyde for overnight, then was dehydrated at −50 °C in lyophilizate (Lyophilizer, Christ Alpha 1–2 LD plus, Germany). Following the coating with gold- palladium (40:60), the sample was examined using scanning electron microscope (SEM, JEOL,SEM 1200 EX, Tokyo,Japan). 2.3.3. Surface area Porosity of the PHEMA cryogel monolith was determined via N2 gas sorption technique by Flowsorb II (Micromeritics Instrument Corporation, Norcross, GA). The specific surface area was determined by Brunauer–Emmett–Teller (BET) method using multipoint analysis and a flowsorb II 2300 from Micromeritics Instrument Corporation, Norcross, GA. 0.5 g of PHEMA cryogel monolith was placed in the sample holder of BET and degassed in a N2-gas stream at 150 °C for 1 h. The adsorption of the N2 gas was performed at 210 °C and the desorption was performed at room temperature. Data obtained from desorption step was used for calculation of specific surface area. 2.4. CNBr activation and HSA immobilization The cryogel monolith samples were activated by CNBr. A series of CNBr solutions (5–80 mg/mL) were prepared, and pH of solutions was adjusted to 11.5 by 2.0 M NaOH. The CNBr solution was passed through cryogel monolithic column for 60 min at 4 °C. The excess reagent was removed by 100 mL of 0.1 M NaHCO3. Then, 50 mL of FeCl3 (5%, w/v) and 1.0 M ethanol amine (pH: 9.1) solutions were passed through the column for 1 h to block the active groups (isourea etc.) on surface. The activated columns were repeatedly washed with 0.5 M NaCl, and stored at 4 °C. HSA immobilization was investigated over the 4.0–8.0 pH range, 0.1–3.0 mg/mL for HSA concentration, 0.5–2.0 mL/min for flow-rate, and 1–120 min for interaction time in continuous mode. The concentration of HSA was determined by absorbance measurements at 280 nm in a UV–Visible spectrophotometer (UV mini-1240, Shimadzu, Tokyo, Japan), using a previously formed calibration curve. The amount of immobilized HSA was determined using the following equation: q ¼ ðCi −C f ÞV=m where Ci and Cf denote initial and final HSA concentrations (mg/mL); V volume of solution (mL); m, mass of cryogel, and q, amount of immobilized HSA per mass of cryogel (mg/g). 2.5. Blood compatibility tests 2.5.1. CT (coagulation time) The cryogel monolith sample was equilibrated with phosphate buffer (0.1 M, pH 7.4) for 24 h at room temperature, then was washed with 0.5 M NaCl, and deionized water. 0.1 mL of frozen human plasma was heated to 37 °C for 2 min and then was interacted with cryogel monolith sample. The coagulation time (CT) was measured by fibrometer method [30]. 2.5.2. APTT (active partial thromboplastin time) and PT (prothrombin time) Following the pretreatment of cryogel with buffer, NaCl, and deionized water, it was interacted with a mixture containing 0.1 mL of human plasma sample (preheated to 37 °C for 2 min) and 0.3 mL of partial thromboplastin (bio Merie-UX, Marcy-I' Etoile, France). After 30 s, CaCl2 (0.1 mL, 0.025 M) was added. Active Partial Thromboplastin

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Time (APTT) and (Prothrombin Time) PT were determined by fibrometer method [31,32]. 2.5.3. Cell adhesion studies Platelets and Leukocytes Counting: The cryogel sample was washed with buffer solution containing 0.1 M KCl to prevent interferences, then was interacted with blood for 1 h. The counting was performed at start of the procedure and when it was ended, using a microscope. 2.5.4. Bilirubin removal from human plasma The freshly frozen human plasma was obtained from Hacettepe University Hospital. The plasma was overloaded with bilirubin equivalent to a level of a hyperbilirubinemia patient, diluted in several ratios, and was interacted with HSA-P[HEMA] cryogel monolith in continuous mode. Bilirubin concentration in plasma was determined by Roche Cobas Integra 400 analyzer (Switzerland). Interaction time, temperature, and bilirubin concentration parameters were investigated to optimize the adsorption process for cryogel samples containing different amounts of HSA. The experiments were conducted in dark. The experiments and measurements were performed in triplicate. 2.6. Desorption and reusability The desorption was investigated in continuous mode for 1 h using 1.0 M NaCl and 0.1 M NaOH containing solution as desorbing agent. The desorption ratio was reported as the percentage of adsorbed amount onto cryogel, using the following equation: Desorption of ratio(%) = [(Desorbed amount of bilirubin/Adsorbed amount of bilirubin)] × 100 The reusability was tested for 10 consecutive adsorption–desorption cycles using the same cryogel sample. The sample was regenerated with 50 mM NaOH, and washed with deionized water between successive cycles. 3. Results and discussion 3.1. Characterization of cryogel The p(HEMA) cryogel monolith is a crosslinked hydrophilic matrix.The equilibrium swelling degree was 7.86 g H2O/g cryogel. Determining the masses of squeezed gel and swollen gel samples, porosity was determined as 69.6%. Due to the elasticity, water in large pores could be squeezed easily from the swollen membranes. The specific surface area determined by multipoint BET analysis was 27.2 m2/g. The sample contained interconnected macropores (20–245 Å), the average diameter being 68.2 Å. The cryogels with high porosity are suitable for use as chromatographic material [33–35]. Moreover, it has a very low flow resistance in chromatographic approach due to their supermacroporosity and interconnected pore-structure [14,36]. The thin polymeric walls and interconnected macropores are easily seen in SEM micrograph (Fig. 1). A biomaterial should be examined by blood and tissue compatibity, mechanical stability, sitotoxicity, biodegradibility tests etc. before use in medical applications. The nature and amount of protein molecules adsorbed on surface of biomaterial determine the subsequent coagulation steps (through intrinsic path) and complement activation (through both intrinsic and extrinsic paths) [37]. APTT test is a measure of activation of intrinsic coagulation factors on surface of biomaterial while PT is a measure of activation of extrinsic coagulation factors and CT measures in-vitro coagulation time. APTT, PT, and CT measurements were performed (Table 1). Compared to control plasma, the decrease in APTT, PT, and CT was not significant and tolerable by the body [38]. Consequently, it can be considered that the blood application of P[HEMA] and HSA-P[HEMA] becomes acceptable with reproducible clotting times with regards to literature values [37]. Platelet and lekocyte

Fig. 1. SEM image of P[HEMA] cryogel.

countings (cell adhesion studies) were also performed (Table 2). The platelet and leukocyte losses were 2.32% and 5.17%, respectively. The changes in specified values were tolerable by body, proving the blood compatibility of HSA-P[HEMA]. Therefore, the P[HEMA] cryogel monolith is as an alternative sorbent to be used in hemoperfusion columns for bilirubin removal from serum. 3.2. HSA immobilization The activation agent (CNBr) reacts with the hydroxyl groups on HEMA to form cyanate esters and imido-carbamates. These groups react with primary amines of proteins. The amine groups yield isourea derivatives with ester and substituted imidocarbamates with cyclic imido-carbamates. The cryogel monolith samples were activated with different amounts of CNBr (5–80 mg/mL). The activated samples were interacted with HSA solutions (0.5 mg/mL) in pH 7.0 phosphate buffer. The amount of HSA attached to samples increased with increasing CNBr concentration up to 60 mg/mL due to the increase in number of active sites on surface and then remained constant (Fig. 2). The optimum CNBr concentration was 60 mg/mL and was kept constant in further runs. The effect of pH on HSA immobilization was examined for 4.0–8.0 range (Fig. 3). The optimum pH was 7.0 for which the immobilized HSA amount was 4.35 mg/g. pK for isourea derivative was nearly 9.5, therefore the activated matrix was positively charged at neutral pH. Since HSA (pI: 4.7) was negatively charged at neutral pH, an electrostatic

Table 1 Coagulation times. Experiments

APTT (s)

PT (s)

CT (s)

Control plasma PHEMA PHEMA-HSA

32.5 32.4 32.3

17.2 16.8 16.9

270 262 267

Each result is the average of three parallel studies.

Table 2 Platelet and leukocyte adhesion on cryogels. Platelet (×10−3/mm3)

Leukocyte (×10−3/mm3)

Initial/final

Loss (%)

Initial/final

Loss (%)

430/400 430/420

6.98 2.32

5.8/5.3 5.8/5.5

8.62 5.17

Substance PHEMA P[HEMA]-HSA

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Fig. 2. Effect of CNBr concentration on immobilization of HSA. HSA concentration: 0.5 mg/mL; flow rate: 0.75 mL/min; pH: 7; T: 25 °C.

interaction occurred, so the optimum pH was reasonable. The proper conformation of protein at the reported pH may also contribute to this favorable interaction. Since the time for interaction of HSA with active sites decreased with an increase in flow rate, a decrease in immobilized amount of HSA was recorded when the flow rate was increased from 0.2 mL/min to 2.0 mL/min (Fig. 4). 0.75 mL/min was chosen as the optimum value due to the small change in capacity values for 0.5 mL/min and 0.75 mL/min. Equilibrium for process was reached in nearly 30 min. (Fig. 5). The initial steep rise in curve was a measure of high affinity of activated cryogel for HSA. The effect of HSA concentration was investigated over the range of 0.1–2.0 mg/mL and the amount of immobilized HSA increased with an increase in HSA concentration (Fig. 6), reaching an equilibrium value of 12.40 mg/g indicating the complete coverage of accessible active sites when HSA concentration was 1.0 mg/mL. P[HEMA] cryogels with different immobilized HSA were encoded as P[HEMA]-HSA1, P[HEMA]-HSA2, P[HEMA]-HSA3, P[HEMA]-HSA4 and P[HEMA]-HSA5. The nonspecific adsorption of HSA for nonactivated matrix was negligible (0.74 mg/g).

Fig. 4. Effect of flow rate on HSA immobilization. HSA concentration: 0.5 mg/mL; pH: 7; T: 25 °C; t:1.0 h.

were interacted with plasma samples for 2 h at room temperature. The nonspecific bilirubin adsorption was 2.46 mg/g. Following the 2 h interaction time, the bilirubin concentration was below the allowable limit of bilirubin in plasma (Fig. 7A). As seen in Fig. 7B, when the amount

3.3. Bilirubin removal from human plasma The plasma samples were overloaded with bilirubin till bilirubin concentration was 3.0 mg/dL. The HSA-P[HEMA] cryogel monoliths

Fig. 3. Effect of pH on HSA immobilization. HSA concentration: 0.5 mg/mL; flow rate: 0.75 mL/min; T: 25 °C; t:1.0 h.

Fig. 5. Effect of incubation time on HSA immobilization. HSA concentration: 1.0 mg/mL; flow rate: 0.75 mL/min; pH: 7; T: 25 °C.

Fig. 6. Effect of HSA concentration on HSA immobilization. Flow rate: 0.75 mL/min; pH: 7; T: 25 °C; t: 30 min.

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Fig. 7. Bilirubin removal performance (A) and adsorption capacities (B) of HSA-P[HEMA]* and P[HEMA] cryogels. Flow rate: 0.75 mL/min; T: 25 °C. * PHEMA-HSA: Different amounts of HSA immobilized P[HEMA] cryogels.

of immobilized HSA on cryogels increased, the amount of adsorbed bilirubin also increased, as expected. The maximum capacity was achieved with P[HEMA]-HSA5 encoded cryogel and P[HEMA]-HSA5 samples were also treated with plasma samples diluted in ratios of 1/2, 1/4, 1/8, and 1/16 for 1 h at room temperature. The decrease in adsorption capacity with a decrease in concentration was due to the change in concentration difference between solution and the cryogel surface [39]. The concentration difference between solution and the cryogel surface increases at the higher concentration, which drives the HSA molecules onto the cryogel surface whilst as the plasma was diluted the concentration of competitors, the low concentration components of plasma, were insufficient to replace bilirubin.The maximum adsorption capacities were obtained as 3.28 mg/g and 22.67 mg/g for P[HEMA] and P[HEMA]-HSA5, respectively (Fig. 8). The temperature effect was analyzed for three different values, the maximum value being 25.4 mg/g at 37.5 °C (Fig. 9). The increase in temperature caused cis to trans conformational change for bilirubin

Fig. 8. Effect of bilirubin concentration on adsorption capacities of P[HEMA]-HSA5 and P[HEMA] cryogels. Flow rate: 0.75 mL/min; T: 25 °C; t: 60 min.

thus reducing the steric hindrance for adsorption [40,41]. Intramolecular hydrogen bonds in bilirubin were weaker at higher temperatures, causing an increase in solubility thus resulting an increase in adsorption capacity. 3.4. Adsorption isotherms The relation between adsorbate concentration in solution and amount of adsorbate on adsorbent in equilibrium was studied by two models, Langmuir and Freundlich [42]. The related equations were used in their linearized forms. The equation 1=Q e ¼ 1=ðQ max :bÞð1=Ce Þ þ 1=Q max : where b is Langmuir constant (mL/mg), Qmax the maximum adsorption capacity (mg/g), Qe equilibrium capacity (mg/g) and Ce, the equilibrium

Fig. 9. Effect of temperature on adsorption capacities of P[HEMA]-HSA5 and P[HEMA] cryogels. Bilirubin concentration in serum: 3.0 mg/dL; flow rate: 0.75 mL/min; T: 25 °C; t: 60 min.

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Table 3 Langmuir and Freundlich constants for adsorption of bilirubin on P[HEMA]-HSA5 cryogel. Experimental

Langmuir

Qmax (mg/g)

Qmax (mg/g)

b (mg/dL)

R2

Kf

Freundlich 1/n

R2

22.67

23.20

20.52

0.9953

17.96

0.23

0.4658

bilirubin concentration (mg/mL) in solution, was used to test the usability of Langmuir isotherm. Ln Q e ¼ ln K f þ ð1=nÞlnCe was adopted for Freundlich isotherm, where Kf and n are Freundlich constants related to adsorption capacity and adsorption intensity. These isotherms constants were given in Table 3. As seen in Table 3, the value of the correlation coefficient (R2) for Langmuir model was very high, near to 1 and the experimental Qmax value (22.67mg/g) was close to calculated Qmax (23.20mg/g) from Langmuir model. According to these results, it was concluded that the Langmuir model was more suitable to explain the adsorption mechanism of bilirubin on the surface of HSA immobilized P[HEMA] cryogel matrix and the Langmuir model assumes that monolayer adsorption, energetically equivalent binding sites, each adsorbate molecule occupies only one site and no lateral interactions between adsorbed molecules to explain adsorption process. 3.5. Adsorption kinetics Mechanism which controls the adsorption process is mass transfer or chemical interaction. Pseudo first-order and second-order kinetic models were applied to experimental data to determine the mechanism. The linearity of log qe vs. t plot, based on the following equation logðqe −qt Þ ¼ logqe −k1 t=2:303 is an indication of Lagergren's pseudo-first order kinetics, diffusioncontrolled mechanism, where k1 is rate constant (min− 1), qe and qt denote adsorbed bilirubin (mg/g) at equilibrium and at any time, t. ðt=qt Þ ¼ 1=k2 qe 2 þ ð1=qe Þt equation was treated to test the applicability for pseudo second-order kinetics, where k2 is the rate constant (g/mg.min.) [43]. Pseudo-first order and p seudo-second order kinetic models were applied to experimental data and the kinetic parameters were given in Table 4. As seen in the Table 4, the pseudo-second order kinetic model is suitable to explain the mechanism of adsorption process according to correlation coefficient (R2) and adsorption capacity (qe). As expected, the kinetic model fitted the data for each cryogel sample due to same adsorption mechanism via HSA-bilirubin interaction. The pseudo second-order kinetics was followed, indicating the chemical interaction of analyte and ligand, without any diffusion limitations.

Fig. 10. The reusability of P[HEMA]-HSA5 cryogel. Bilirubin concentration in serum: 3.0 mg/dL; flow rate: 0.75 mL/min; T: 25 °C; t: 60 min.

3.6. Desorption and reusability The desorption agent was 1.0 M NaOH and 1.0 M NaCl containing solution. 20 mL desorption agent was passed through the column for 1 h, giving a value of desorption ratio higher than 96%, in all runs. The decrease in adsorption capacity was 4% following the 10 consequtive adsorption–desorption cycles (Fig. 10) proving the reusability of the adsorbent.

4. Conclusion Considering the disadvantages of conventional treatment methods for hyperbilirubinemia, the synthesis of an efficient adsorbent for hemoperfusion was targeted. Due to the high affinity between HSA and bilirubin, HSA was chosen as ligand, and immobilized onto the HEMA based matrix following CNBr activation. The adsorbent was synthesized as a cryogel monolith. The optimum immobilization parameters were obtained at 60 mg/mL CNBr concentration at 1.0 mg/mL HSA, pH 7.4 and 0.75 mL min− 1 flow rate for 30 min of interaction time. The effect of HSA contents, incubation time, bilirubin concentration in plasma and temperature was investigated for bilirubin removal performance of HSA immobilized p[HEMA] cryogel monolith. The optimum conditions were determined as 12.40 mg HSA/g cryogel, 3.0 mg/dL bilirubin concentration and 37.0 °C for 60 min of interaction period and the maximum adsorption capacity was as 25.4 mg/g under these conditions. As a conclusion, this novel adsorbent had a higher adsorption capacity with high specificity for bilirubin removal than other adsorbents [23,27,44,45]. Moreover, in terms of ease in synthesis, biocompatibility, advantages of cryogel format in continuous mode, inexpensiveness, reusability, the resulting matrix modified with HSA was a suitable solid-phase extraction sorbent to be used in hemoperfusion columns.

Table 4 Pseudo-first order and pseudo-second order kinetic parameters for adsorption of bilirubin on P[HEMA]-HSA cryogels at 3.0 mg/dL bilirubin concentration. Experimental

Pseudo-first order kinetic

Cryogels

Qeq (mg/g)

k1 (min−1)

qeq (mg/g)

R2

k2 (g/mg.min)

Pseudo-second order kinetic qeq (mg/g)

R2

P[HEMA]-HSA1 P[HEMA]-HSA2 P[HEMA]-HSA3 P[HEMA]-HSA4 P[HEMA]-HSA5

13.32 16.32 18.72 21.54 22.67

0.015 0.017 0.019 0.026 0.030

20.76 19.53 18.82 16.44 14.98

0.4839 0.5886 0.7024 0.8238 0.7924

0.050 0.087 0.093 0.102 0.131

16.13 17.99 20.24 23.47 24.39

0.9928 0.9987 0.9961 0.9956 0.9954

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