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Molecularly imprinted composite cryogel for extracorporeal removal of uric acid
Colloids and Surfaces B: Biointerfaces 183 (2019) 110456
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Molecularly imprinted composite cryogel for extracorporeal removal of uric acid
T
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Bilgen Osmana, , Engin Sagdilekb, Merve Gümrükçüa, Aslı Göçenoğlu Sarıkayaa a b
Bursa Uludag University, Department of Chemistry, Bursa, Turkey Bursa Uludag University, Department of Biophysics, Bursa, Turkey
A R T I C LE I N FO
A B S T R A C T
Keywords: Uric acid Extracorporeal treatment Cryogel Haemocompatibility Removal
In this study, uric acid (UA)-imprinted poly (hydroxyethyl methacrylate-N-methacryloyl-amido-L-cysteine methyl ester)-Fe3+ [poly(HEMA-MAC)-Fe3+] nanoparticle-embedded poly(acrylamide-methyl methacrylate) cryogel [p(AAm-MMA)-MIP] was synthesized for selective UA adsorption. The nanoparticles were prepared via molecular imprinting. The prepared p(AAm-MMA)-MIP cryogel was characterized by Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and swelling test. The swelling degree of the p(AAmMMA)-MIP cryogel was determined as to 7.56 g H2O/g cryogel. The prepared cryogel was used for UA adsorption from aqueous solution.The effects of pH (4.0–8.0), initial UA concentration (5–40 mg/L), temperature (4 °C, 25 °C and 35 °C) and contact time on the UA adsorption capacity were detailedly investigated. UA adsorption data were applied to Langmuir and Freundlich isotherm models. The adsorption data were well fitted with pseudo-second order kinetic model. The thermodynamic parameters (ΔG ͦ , ΔH ͦ , ΔSo) demonstrated that the adsorption process was endothermic and spotaneous at 4 °C, 25 °C and 35 °C. The cryogel was also used for UA adsorption from human serum. The effects of the composite cryogel treatment on blood cells and hemostatic parameters were evaluated by using hemogram analyses, coagulation studies, thromboelastography and platelet aggregation studies. The results showed that the cryogel treatment has an allowable effect on blood cell counts and hemostatic parameters demonstrating the applicability of prepared composite cryogel for UA removal from human serum.
1. Introduction Uremic syndrome is the terminal clinical manifestation of kidney failure [1]. The metabolic compounds refered to uremic toxins are retained in the body during uremic sendrom and adversely affect the biological functions. Uric acid (UA), a water-soluble uremic toxin, is the end product of human purine-nucleotide metabolism. The physiological concentration of UA in blood plasma varies with age and gender, ranging from 2.0 mg/dL to 6.8 mg/dL [2]. UA levels above these limits cause hyperuricemia. Hyperuricemia is associated with numerous diseases, such as Lesch-Nyan syndrome, gout, cardiovascular diseases and kidney diseases [3,4]. Therefore, decreasing serum UA levels has been proposed as a priority in treating hyperuricemia [5]. The level of uremic toxins generally decreases via haemodialysis (HD) or by using therapeutic enzymes that metabolize these harmful biological compounds [6,7]. However, the UA levels have been reported to not sufficiently decrease in uremic patients even with adequate dialysis. Moreover, increasing the adequacy or duration of dialysis is not
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effective [8]. Therapeutic enzymes can be used intravenously to decrease UA levels, but immunity reduces their half-life in circulation when they are used repeatedly for a long time. Hemoperfusion is an extracorporeal treatment that removes endogenous and exogenous toxic substances from patients’ blood. It has been mainly used for poisonings, hepatic failures and treating uraemia and its complications. Affinity adsorption is a proper alternative to conventional hemoperfusion for removing any undesired substances from the patient’s serum in different pathologies. [9,10]. However, developing affinity sorbents for blood treatment is one of the most important problems because these adsorbents should be highly selective and have a relatively high adsorption capacity for the target toxin. Moreover, they should be reusable, biocompatible and mechanically resistant [11]. Many modified polymeric resins meet these requirements for UA removal [12–14]. However, the adsorption capacities of the resins are low, and, more importantly, they have no selectivity for UA. Molecularly imprinted polymers (MIPs) have a high selectivity for
Corresponding author. E-mail address: [email protected] (B. Osman).
https://doi.org/10.1016/j.colsurfb.2019.110456 Received 3 June 2019; Received in revised form 11 August 2019; Accepted 22 August 2019 Available online 23 August 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
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using the procedure developed in our previous study [26]. A detailed experimental procedure for nanoparticle synthesis are given in the Supporting Information. The following procedure was utilized for synthesis of UA-imprinted PHEMA-MAC-Fe3+ nanoparticle-embedded cryogel [p(AAm-MMA)-MIP]: 0.285 g of N,N′ methylenebis(acrylamide) was dissolved in 10 mL of deionized water. Then, 0.71 g of acrylamide (AAm) and 1.07 mL of methyl methacrylate (MMA) were mixed with an aqueous solution of the UA-imprinted nanoparticles. The prepared solutions were mixed together, and then APS and TEMED were added to the mixture at 0 °C. The mixture was poured into a cold syringe prior to polymerization at −18 °C for 18 h. The p(AAm-MMA)MIP cryogel was excessively washed with deionized water after the cryogel thawed to room temperature. The non-imprinted PHEMA-MACFe3+ nanoparticle-embedded cryogel [p(AAm-MMA)-NIP] was prepared via same procedure with the addition of the non-imprinted PHEMA-MAC-Fe3+ nanoparticles prepared without adding UA. The amount of PHEMA-MAC-Fe3+ nanoparticles (MIP or NIP) in the cryogel was 0.57 mg. FTIR analyses were conducted by Spectrum 100 model FTIR spectrometer (Perkin Elmer, USA). The physical morphology of the cryogels coated with thin layer of gold (100 Å) was monitored via a scanning electron microscope (Carl Zeiss Evo 40, Germany). The swelling degree of the p(AAm-MMA)-MIP cryogel was determined in deionized water. The cryogel was incubated in 200 mL of deionized water at room temperature for 2 h. The cryogel sample was taken from the water and carefully weighed after wiping with filter paper. After that, the cryogel was dried at 40 ͦ C for 24 h and weighed. The swelling degree was calculated using Eq. (1);
their target molecules [15] and have been effectively used as affinity adsorbents to remove toxic compounds from blood [16,17]. MIPs with UA recognition have also been reported in recent years. Cristallini et al., 2004 [18] and Silvestri et al., 2006 [19] prepared acrylic acid‐acrylonitrile copolymer membranes to selectively remove UA from the blood. Functional polyamidoxime/crosslinked polyvinyl alcohol (PAO/ CPVA) microspheres with excellent binding affinity for UA were prepared by Gao et al., 2010 [20]. The polymers based on ethylene glycol dimethacrylate (EGDMA) and dimethylaminoethyl methacrylate (DMAEMA) were also synthesized for selective UA adsorption [21,22]. Today, the use of cryogels that present relatively low flow resistance due to their interconnected macroporous structures has attracted attention in biomedical applications. They have a high mechanical strength and a resistance to chemicals. Composite cryogels, including MIP particles, have also been used for affinity adsorption of toxic compounds from blood. The ion-imprinted beads embedded poly (hydroxyethyl methacrylate) (PHEMA) cryogel was successful for the in vitro removal of iron (III) ions from beta-thalassemic human plasma [23]. Baydemir et al., 2009 [16] prepared a supermacroporous PHEMA cryogel embedded with bilirubin-imprinted particles. It is possible to obtain both low back pressure and selectivity by embedding MIPs in the cryogels, which facilitates separation processes. Zheng et al, 2012 [24] prepared poly (vinyl alcohol) cryogels embedded with activated carbon microparticles to investigate the potential use of cryogelation technology as an activated carbon-anchoring backbone in new hemoperfusion-based applications. Although the targeted uremic toxins were removed effectively, the haemocompatibility of the composite cryogel was very low due to significant decreases in red blood cell (RBC) counts (80%), white blood cell (WBC) counts (> 50%) and platelet (PLT) counts (> 50%). [25]. In this study, a composite cryogel including selective nanoparticles was prepared for UA removal for the first time. The haemocompatibility of the composite cryogel was tested by hemogram analyses, coagulation studies, thromboelastography and platelet aggregation studies. UAimprinted poly (hydroxyethyl methacrylate-N-methacryloyl-amido-Lcysteine methyl ester)-Fe3+ [PHEMA-MAC-Fe3+] nanoparticles were prepared via molecular imprinting. The UA-imprinted PHEMA-MACFe3+ nanoparticles were embedded into poly(acrylamide-methyl methacrylate) [p(AAm-MMA)] cryogel to prepare the composite cryogel [p (AAm-MMA)-MIP]. Adsorption studies were conducted to evaluate the effects of pH, initial UA concentration, temperature and contact time on the adsorbed amount of UA onto the p(AAm-MMA)-MIP cryogel. The adsorption data were used for isothermal, kinetic and thermodynamic analyses. The UA removal performance of the p(AAm-MMA)-MIP cryogel from human plasma was also evaluated together with other blood components.
Swelling degree =
ws − w0 w0
(1)
where ws and w0 were the weight of swelled and dry cryogels, respectively. Macroporosity of the synthesized cryogel was calculated according to Eq. (2). In the procedure, cryogels were first swelled up to equilibrium and weighed (ws ); then squeezed by hand and weighed (wsq ); and finally dried until the constant weight (wd ) obtained. Macroporosity was calculated through following equation:
Macroporosity % =
(ws ) − (wsq ) wd
x100
(2)
2.3. Adsorption experiments UA adsorption experiments were conducted on the basis of continious analysis. UA solutions were circulated through the p(AAmMMA)-MIP cryogel column via a peristaltic pump (Multi channel cassette pumps–205S/CA, Watson Marlow). The cryogel column (bed height: 3 cm, column diameter: 1.4 cm) was allowed to equilibrate with the UA solutions for 8 h at a flow rate of 0.5 mL/min. Then, the absorbances of UA solutions were measured at 286 nm wavelength by UV–vis spectrophotometer (UV-1700, Shimadzu). Eq. (3) was used to calculate the adsorbed amount of UA;
2. Experimental 2.1. Materials Uric acid (C5H4N4O3), ammonium persulfate (APS), methacryloyl chloride and L-cysteine methyl ester hydrochloride were purchased from Sigma (St. Louis, USA). Acrylamide (AAm), hydroxyethyl methacrylate (HEMA) and ethylene glycol dimethacrylate (EGDMA) were purchased from Fluka. N,N,N′,N′-tetramethylethylenediamine (TEMED) and N,N′-methylenebis(acrylamide) (MBAAm) were supplied from Merck. Disposable cups and pins (Clear) and 0.2 M CaCl2 were purchased from Haemonetics Corporation. Adenosine diphosphate (ADP), collagen, and epinephrine were purchased from Bio/Data Corporation (PAR/PAK®II). Elga Flex3 water purification system (Veloia Water Solutions & Technologies, France) was used to prepare purified water.
Q=
(C0 − C ) V m
(3)
where Q is the amount of adsorbed UA (mg/g); Co and C are the initial and final concentrations of UA in aqueous solution, respectively (mg/ L). m is the mass of the nanoparticles used (g) and V is the volume of the aqueous phase (L). To determine the influence of the aqueous phase pH on UA adsorption capacity of the p(AAm-MMA)-MIP cryogel, the initial pHs of the UA solutions were varied in the range of 4.0-8.0. The initial concentration of UA was 5 mg/L. The effect of initial UA concentration was investigated in the concentration range of 5–40 mg/L at pH 6.0. The kinetics of UA adsorption were analysed for 480 min (pH 6.0, initial UA
2.2. Synthesis and characterization of p(AAm-MMA)-MIP cryogel UA-imprinted PHEMA-MAC-Fe3+ nanoparticles were prepared by 2
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concentration: 30 mg/L) at 4 °C, 25 °C and 35 °C.
2.6. Adsorption studies in human serum
2.4. Selectivity and reusability studies
The p(AAm-MMA)-MIP and p(AAm-MMA)-NIP cryogels were used for UA adsorption from human plasma. A blood sample was collected into a tube that included gel. Then, the sample was centrifugated at 5000 g for 15 min to remove blood cells. An undiluted sample and diluted samples (with ratios of 1:2 and 1:8) spiked with 35 mg/L of UA were prepared, for a total volume of 7 mL.The prepared samples were separately passed from p(AAm-MMA)-MIP and p(AAm-MMA)-NIP cryogels for 4 h (flow rate:0.5 mL/min). The removal of blood components (uric acid, urea, creatinine, glucose, total cholesterol, triglyceride, total protein, albumin, Na+, K+, Cl−, Ca2+) was also investigated by interacting 4 mL of the undiluted serum sample with the p(AAm-MMA)-MIP, p(AAm-MMA)-NIP and p (AAm-MMA) cryogels. The initial UA concentration of the blood sample was 85 mg/L. The concentrations of blood components in the treated and untreated serum samples were determined with ARCHITECT® c 16000TM (Abbott Laboratories, Diagnostic Division, Abbott Park, IL, USA). The experiments were repeated three times. The removal percentage was calculated according to Eq. (4).
The experimental procedures for selectivity and reusability studies of the p(AAm-MMA)-MIP cryogel are given in the Supporting Information. 2.5. Blood compatibility studies Procedure of blood collection and statistical analysis methods used for data evaluation are given in the Supporting Information. To avoid platelet activation and artificial hemolysis a syringe (Hamilton) rather than a peristaltic pump was used for a single-pass blood perfusion experiment through the cryogel in blood compatibility studies. 2.5.1. Hemogram analysis Blood samples (4 mL) taken from seven healthy volunteers were collected into tubes that contained EDTA. Firstly, the p(AAm-MMA)MIP cryogel was swollen by saline solution (% 0.9 NaCl, 5 mL). Then, 2 mL of the blood sample was passed from the p(AAm-MMA)-MIP cryogel. The remaining 2 mL of the sample was kept at room temperature as an untreated (control) sample. Hemogram analyses of the samples were performed by CELL-DYN®RubyTM hemogram device (Abbott Laboratories, Diagnostic Division, Abbott Park, IL, USA). The hemogram parameters used for evaluation are given in the Supporting Information.
Removal % =
(CO − C ) X 100 CO
(4)
where CO and C are the initial and final concentrations of serum components before and after the cryogel treatment, respectively. 3. Results and discussion 3.1. Properties of p(AAm-MMA)-MIP composite cryogel
2.5.2. Coagulation tests Blood samples (4 mL) were collected from the seven healthy volunteers whose bloods were also used in the hemogram analyses. The blood samples were collected into tubes that included sodium sitrate (1:9 v/v). After that, 2 mL of the blood sample was passed from the p (AAm-MMA)-MIP cryogel swollen by saline solution (% 0.9 NaCl, 5 mL). The remaining 2 mL of the sample was kept at room temperature as an untreated (control) sample. The prothrombin time (PT), activated partial thromboplastin time (APTT) and the international normalized ratio (INR) for treated and untreated blood samples were automatically generated with BCS® XP System (Marburg, Germany).
The p(AAm-MMA)-MIP cryogel was synthesized by polymerization of AAm, methyl MMA and MBAAm in the presence of UA-imprinted PHEMA-MAC-Fe3+ nanoparticles. Firstly, the composite cryogel was characterized by FTIR analysis. In the FTIR spectrum (Fig. SI1), a broad absorption band arising from NeH stretching was observed around 3300 cm−1. The strong adsorption bands observed at 1655 cm−1 and 1723 cm−1 belong to stretching vibration of amide carbonyl group and ester carbonyl group, respectively. The results showed that the applied polymerization procedure enabled the synthesis of the p(AAm-MMA)MIP cryogel. The swelling degree and macroporosity of the composite cryogel were determined as 7.56 g H2O/g cryogel and 68.27%, respectively. The main purpose of embedding the UA-imprinted PHEMA-MACFe3+ nanoparticles into the cryogel is to take advantage of the cryogel’s superior flow characteristics. The physical morphologies of the p(AAmMMA), the p(AAm-MMA)-MIP and the p(AAm-MMA)-NIP cryogels were monitored by SEM analyses. The SEM photographs with different magnifications are shown in Fig. 1. The p(AAm-MMA) non-embedded cryogel has interconnected macropores (Fig. 1a and b). The surfaces of the macropores were also completely covered with PHEMA-MAC-Fe3+ nanoparticles (Fig. 1c–e). SEM analyses showed that the applied polymerization procedure enabled the preparation of a UA-selective p(AAm-MMA)-MIP composite cryogel. Thus, the interconnected macropores make it possible to take advantage of the cryogel’s relatively low flow resistance.
2.5.3. Thromboelastography Thromboelastogram (TEG) measurements were performed with TEG®5000 Thrombelastograph® Hemostasis System (HAEMONETICS ORPORATION, MA, USA). Blood samples were put into vacutainer tubes that included 0.9 mL 0.106 M sodium citrate as an anticoagulant (citrate-blood ratio, 1:9 v/v). The volume of blood sample (3 tubes) collected from each volunteer was 27 mL. 4 mL of the blood sample was used for TEG measurements. The remaining 23 mL of the sample was kept for aggregation studies. A detailed procedure of TEG measurements is given in the Supporting Information. 2.5.4. Aggregation measurements An optical aggregometer (Platelet Aggregation Profiler PAP-4CD®, Bio/Data Corporation, Montgomery, PA, USA) was used to perform aggregation measurements. The blood samples (23 mL) anticoagulated with sodium citrate (citrate-blood ratio, 1:9 vol/vol) were centrifugated for 10 min at 250gto prepare platelet-rich plasma (PRP). The plateletpoor plasma (PPP) was prepared by centrifugation of the remaining blood at 2000gfor 15 min. The obtained PRP (5 mL) were divided into two parts. The 2.5 mL of the PRP sample was passed from the p(AAmMMA)-MIP cryogel. The remaining 2.5 mL of the PRP sample was kept under room temperature as an untreated (control) sample. The treated and untreated PRP samples were simultaneously studied in the two channels of the aggregometer. A detailed procedure of aggregation measurements is given in the Supporting Information.
3.2. Adsorption studies 3.2.1. pH effect pH is an effective parameter because UA is adsorbed onto the embedded UA-imprinted PHEMA-MAC-Fe3+ nanoparticles via metal-chelate interaction of MAC, Fe3+ and UA. The potentiometric titration studies in our previous study [26] showed that MAC-Fe3+-UA complex forms at pH 4.0 and is stable at pH values lower than 8.0 (Fig SI2). In the pH range of 4.0–8.0, UA reversibly interacts with the MAC-Fe3+ complex depending on the amount of H+ ions in the medium, i.e pH. 3
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Fig. 1. SEM photographs of the p(AAm-MMA) cryogel (a and b); the p(AAm-MMA)-MIP cryogel (c, d and e); the UA-imprinted PHEMA-MAC-Fe3+ nanoparticles (f) at different magnificaions [(a) 1500×, (b) 3000×, (c) 1520×, (d) 3500×, (e) 500× and (f) 12,000×].
However, MAC-Fe3+ complex distrupts due to the protonation of sulphydryl group (-SH) of the MAC monomer at the pH values lower than 4.0. Additionally, the MAC-Fe3+-UA complex hydrolyzes at the pH values higher than 8.0. Therefore, the effect of pH on the adsorbed amount of UA was investigated in the pH range of 4.0–8.0 (Fig. 2). The adsorbed amount of UA onto the p(AAM-MMA)-MIP cryogel did not significantly change in the studied pH range since the pH of the medium is favorable for MAC-Fe3+−UA complex formation. Thus, pH 6.0 was
determined as an optimal pH for UA adsorption studies.
3.2.2. Isothermal studies The influence of initial UA concentration on UA adsorption was investigated at 4 °C, 25 °C and 35 °C. The concentrations of the UA solutions were varied from 5 to 40 mg / L (pH 6.0). The adsorption experiments could not be performed at higher UA concentrations because the maximum solubility of UA in water is 40 mg/L. The results are 4
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Fig. 2. Effect of pH on UA adsorption onto the p(AAm-MMA)-MIP cryogel (Solution volume: 25 mL, initial UA concentration: 5 mg/L, Temperature: 25 ͦ C, Flow rate: 0.5 mL/min, Time: 8 h).
Fig. 4. Effect of temperature on UA adsorption onto the p(AAm-MMA)-MIP cryogel (Solution volume:25 mL, initial UA concentration: 30 mg/L, Temperature: 4 °C, 25 °C, 35 °C, pH 6.0, Flow rate: 0.5 mL/min, Time: 8 h).
adsorbate coverage is limited to one molecular layer. Therefore, the adsorption data can be fitted by both the Langmuir and the Freundlich isotherm models especially at the low solute concentrations [29]. The UA adsorption data were also fitted with both isotherm models in the low UA concentration range (5–40 mg/L). The UA adsorption capacities of the p(AAm-MMA) and the p(AAmMMA)-NIP cryogels were also evaluated in the same concentration range and compared to that of the p(AAm-MMA)-MIP cryogel at 25 °C (pH 6.0) (Fig. SI3). The adsorption capacity of the p(AAm-MMA)-MIP cryogel was greater than that of the p(AAm-MMA)-NIP cryogel. In addition, the p(AAm-MMA) cryogel has no UA adsorption. The superior UA adsorption performance of the p(AAm-MMA)-MIP cryogel demonstrated that the embedded UA-imprinted PHEMA-MAC-Fe3+ nanoparticles have binding sites with a high affinity for UA.
Fig. 3. Effect of initial UA concentration on UA adsorption onto the p(AAmMMA)-MIP cryogel (Solution volume: 25 mL, pH: 6.0, initial UA concentration: 5–40 mg/L, Temperature: 4 °C, 25 ͦ C,35 °C, Flow rate: 0.5 mL/min, Time: 8 h).
3.2.3. Kinetic studies UA adsorption kinetics investigated at 4 °C, 25 °C and 35 °C are depicted in Fig. 4. The initial UA concentration was 30 mg/L at pH 6.0. The equilibrium time was around 180 min, indicating a fast adsorption rate. Additionally, the amount of adsorbed UA increased with an increasing temperature, indicating the adsorption process’s endothermic nature. The results demonstrated that the prepared cryogel was suitable for rapid and selective UA adsorption. The experimental data were used to examine pseudo-first-order kinetic model [30] and pseudo-second-order kinetic model [31] at three temperatures (277, 298 and 308 K). The linear forms of the applied models are given in Table SI2. The parameters of the kinetic models are summarized in Table SI4. The pseudo-second-order model has the highest correlation coefficients (R2), which are all greater than 0.9997. Moreover, q e (calculated) is close to q e (experimental) suggesting the second-order nature of the adsorption process. The results showed that the rate-limiting step may be the chemical adsorption [32].
provided in Fig. 3. The adsorbed amount of UA increased with an increase in the initial UA concentration at three different temperatures. The maximum amount of UA adsorbed onto the cryogel was determined as to 687.6 mg UA / g at 25 °C (pH 6.0). In addition, the adsorbed amount of UA increased from 574.7 mg / g to 777.9 mg / g with an incease in the temperature from 4 °C to 35 °C. The result indicated that the UA adsorption process was endothermic the in nature. The adsorption capacity of the cryogel was also compared to other UA-imprinted adsorbents (Table SI1). The Langmuir and Freundlich isotherm models were applied to the UA adsorption data with respect to initial UA concentrations from 5 mg/L to 40 mg/L at 4 °C, 25 ͦ C and 35 °C The linear forms of the Langmuir isotherm model [27] and the Freundlich isotherm model were given in Table SI2. The Langmuir isotherm model assumes that the adsorption is monolayer, the adsorbent surface is homogeneous and the energy of the binding sites on the adsorbent surface are equal. The Freundlich isotherm model is used to describe adsorption processes of the adsorbent with heterogeneous binding sites. The energies of the binding sites of the adsorbent are not equal and the interaction between adsorbate molecules is also possible [28]. The calculated parameters for Langmuir and Freundlich isotherm models are shown in Table SI3. Both the Freundlich and the Langmuir isotherm models offered high correlation coefficients for all temperatures and well fitted the adsorption data. The Freundlich isotherm equation is an empirical equation and can be derived from the Langmuir equation by assuming that the binding sites are heterogeneous. The Freundlich isotherm model assumes that the adsorbed amount of solute increases with increasing concentration. Conversely, the Langmuir isotherm supposes that the adsorbed amount of solute reaches a constant value at high concentrations since the extent of
3.2.4. Thermodynamic parameters Thermodynamic parameters; Gibbs free energy change ( ΔGo ), entropy change ( ΔS o ) and enthalpy change ( ΔH o ) for UA adsorption onto p(AAm-MMA)-MIP cryogel were calculated using Van’t Hoff equation and Eq. (6) and are discussed in the Supporting Information (Table SI5).
lnK =
ΔS o ΔH o 1 − ( ) R R T
ΔGo = ΔH o − TΔS o
(5) (6)
3.3. Selectivity and reusability of the p(AAm-MMA)-MIP cryogel The p(AAm-MMA)-MIP cryogel has a high selectivity when theophylline was used as a competitor molecule. The results of selectivity 5
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cryogel also depleted insignificant amount of creatinine and glucose probably because these compounds can act as a ligand to coordinate Fe3+ ions with oxygen and nitrogen atoms in the structure. The removal percent of UA was 34.8% for p(AAm-MMA)-MIP cryogel. Consequently, the p(AAm-MMA)-MIP cryogel with a selectivity for UA has a usage potential as a hemoperfusion adsorbent for UA removal from human blood with allowable effects on other plasma components. 3.5. Effects of the p(AAm-MMA) composite cryogel treatment on blood cells and hemostatic parameters Hemoperfusion is an alternative blood purification procedure in which whole blood is exposed directly to sorbent materials with the capacity to selectively or nonselectively bind endogenous or exogenous toxins. After contact with blood, however, the adsorbent entraps red blood cells, platelets and leukocytes. Therefore, the effects of the p (AAm-MMA)-MIP cryogel treatment on blood cells’ counts were rigorously investigated by hemogram analyses, and the significance of differences between samples was statistically evaluated.. The changes in hemogram parameters that resulted from the cryogel treatment are provided in Table SI7. The significant decrease in HTC counts (8.6%, p < 0.01) after the cryogel treatment reflects the total decrease in white blood cell counts, red blood cell counts and platelet counts. The percentage decreases were 6.5%, 8.3% and 10.7% for WBC, RBC and PLT counts, respectively. Significant decreases were also observed in MONO counts (14%, p < 0.01), probably because they are the largest white blood cells, and in NEU counts (8.6%, p < 0.01) because they are highly abundant WBCs. However, thrombocytopenia, leukopenia, anaemia and a decrease in fibrinogen are the commonly reported adverse reactions to hemoperfusion [33]. For instance, transient leukopenia is commonly observed during hemoperfusion in humans and may result from a complement activation of surface contact, with a margination of leukocytes similar to that observed during haemodialysis. [34]. The decrease in RBC counts was statistically significant (8.3%, p < 0.01). The parameters related to red blood cells, such as HGB (8.8%, p < 0.01), MCV (0.33%, p < 0.01) and RDW (2.43% p < 0.01), were also affected by cryogel treatment. Moreover, the decreases in MCV and RDW counts clearly demonstrated that the large erythrocytes were trapped in the cryogel. The PLT count was also affected by the cryogel treatment, presenting a significant decrease (10.7%) despite platelets being the smallest components of blood. The result probably proceeded from the platelets’ activation and subsequent adherence to the cryogel surface. However, profound platelet depletion was commonly observed in hemoperfusion devices and platelet loss in current hemoperfusion devices is about 30% or less [35]. While the decrease in PLT counts was 10.7% for a single-pass blood perfusion experiment, the decrease in PLT counts was 12.4% when the blood sample interacted with the p(AAm-MMA)-MIP cryogel for 3 h (data not shown). Additionally, no significant decreases were detected in the other hemogram parameters, such as EOS, BASO, LYM, MCH, MCHC and MPV. Consequently, the p(AAm-MMA)-MIP cryogel treatment has an allowable effect on blood cells. The adsorption or activation of coagulation factors is a frequent problem during clinical hemoperfusion. The most significant change occurs in the fibronectin and fibrinogen concentration [36], even if the polymer-coated activated charcoal is used as an adsorbent [37]. Therefore, the p(AAm-MMA)-MIP cryogel treatment’s effect on coagulation parameters was evaluated by measuring the prothrombin time (PT), the international normalized ratio (INR) and the activated partial thromboplastin time (APTT). The PT and APTT times and the INR values presented in Table SI8 clearly show that coagulation factors were not affected by the cryogel treatment because there is no statistically significant change in PT or APTT times or INR values (p > 0.05) after the cryogel treatment. A hemostatic test called thromboelastography measures the
Fig. 5. UA adsorption from human serum samples spiked with 35 mg/L UA (Solution volume: 7 mL, initial UA concentration: 35 mg/L, Temperature: 25 ͦ C, Flow rate: 0.5 mL/min, Time: 4 h, n = 3).
studies are detailedly discussed in the Supporting Information (Fig SI4, Table SI6). The p(AAm-MMA)-MIP composite cryogel was also used for ten successive adsorption/desorption cycles (Fig. SI5). UA interacts with the p(AAm-MMA)-MIP cryogel via metal-chelate interaction with Fe3+ ions. Therefore, the adsorbed UA can be desorbed easily by decreasing the pH of the medium. The result showed that the prepared cryogel can be utilized for UA adsorption without a decrease in UA adsorption performance. 3.4. UA adsorption from human serum Human serum is a complex mixture including electrolytes, antigens, antibodies, hormones and any exogenous substances. The p(AAmMMA)-MIP cryogel was used for UA adsorption from human serum to investigate the cryogel’s selectivity in a real sample. The undiluted serum sample and the diluted (with ratios of 1:2 and 1:8) serum samples, which were spiked with 35 mg/L UA, interacted with the p(AAmMMA)-MIP cryogel. The efficiency of the p(AAm-MMA)-NIP cryogel was also evaluated to show the effectiveness of the molecular imprinting. The results are provided in Fig. 5. The adsorbed amounts of UA onto the p(AAm-MMA)-MIP cryogel were 148.6, 157.1 and 186.6 mg/g for the undiluted sample, the diluted sample in the ratio of 1:2 and the diluted sample in the ratio of 1:8, respectively. The amounts of UA adsorbed onto the p(AAm-MMA)-NIP cryogel were lower than those adsorbed onto the p(AAm-MMA)-MIP cryogel for all studied samples. Although all the serum samples were spiked with 35 mg/L UA, the highest amount of UA adsorption was obtained for the sample diluted at the ratio of 1:8, suggesting that low-concentration plasma components were not sufficient to replace UA. However, the adsorbed amounts of UA calculated for the undiluted sample and the diluted sample in the ratio of 1:2 were not significantly different, indicating that the p(AAm-MMA)-MIP cryogel has a high affinity for UA molecules. Additionally, the percent of removed plasma components, such as Ca2+, Cl−, K+, Na+, creatinine, glucose, albumin, cholesterol, total protein, triglyceride and urea, was investigated to assess the effectiveness and possible side effects that the p(AAm-MMA)-MIP cryogel may improve in hemoperfusion. The percent of removed plasma components for the non-embedded p(AAm-MMA), the p(AAm-MMA)-MIP and the p (AAm-MMA)-NIP cryogel treatments are depicted in Fig. SI6. The results showed that the p(AAm-MMA) cryogel has no adsorption. The p (AAm-MMA)-MIP and p(AAm-MMA)-NIP cryogels did not deplete albumin, cholesterol, total protein and triglyceride. The removal percent of urea was 12.3% for p(AAm-MMA)-NIP cryogel while the p(AAmMMA)-MIP cryogel has no urea adsorption owing to the selectivity of the cryogel. The removal percentages of electrolytes were not greater than 12.7% for p(AAm-MMA)-MIP cryogel. The p(AAm-MMA)-MIP 6
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Acknowledgement
dynamics of clot formation and the stability and strength of clots. The thromboelastogram parameters extend our knowledge about the effect of foreign material contact on hemostatic parameters. Therefore, the TEG parameters of the treated and untreated blood samples were determined and summarized in Table SI8. The graph showing the TEG curves is also depicted in Fig SI7. The results indicate that the cryogel treatment caused statistically significant decreases in maximum amplitude (MA, mm, p < 0.001), clot strength (G, dyn/cm2, p < 0.01) and amplitude at 60 min (A60, mm, p < 0.05)—suggesting that cryogel column treatment decreased clot strength and amplitude. The decrease in platelet counts (10.7%) after the cryogel treatment probably caused a decrease in clot strength because MA (mm) and G (dyn/ cm2) are relate directly with the number and function of the platelets. Other TEG parameters such as angle (α, degrees) and reaction time (R, min) relate to coagulation factors and did not change significantly. These results clearly show that the cryogel treatment did not affect the coagulation parameters, which is consistent with the results of coagulation studies. Platelets mainly function to aggregate and form blood clots at injured vessel walls. Platelets’ contact with foreign-material surfaces results in adhesion and subsequent activation, depending upon the properties of the surface (e.g. its wettability, roughness, polarity and chemical composition) [38]. Surface roughness induces platelet adherence and hydrophilic surfaces prevent cell attachment [39]. Therefore, the aggregation profiles of platelets were also investigated to determine the effect of a cryogel treatment. The changes in maximum aggregation (%) and slope parameters obtained by different aggregation agents (e.g. ADP, epinephrine and collagen) are presented in Table SI8. The aggregation curves for ADP, epinephrine and collagen are also shown in Fig SI8. The p(AAm-MMA)-MIP cryogel treatment caused statistically significant increases in maximum aggregation (%) values for all studied stimulants (p < 0.05). However, the percentage increases in maximum aggregation (%) values were different for ADP (16.7%), collagen (5.3%) and epinephrine (7.5%), probably because the agents have distinct intracellular pathways. Thus, the platelets’ aggregation behaviour was affected by the cryogel treatment because the platelets become proactive.
The authors thank to Research Foundation of Bursa Uludag University for its financial support in the research project of OUAP(F)2016/4. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.110456. References [1] R. Vanholder, R. de Smet, C. Hsu, P. Vogeleere, S. Ringoir, Semin. Nephrol. 14 (3) (1994) 205–218. [2] E.P. de Oliveira, R.C. Burini, Diabetol. Metab. Syndr. 4 (12) (2012) 1–7. [3] F. Jossa, E. Farinaro, S. Panico, V. Krogh, E. Celentano, R. Galasso, M. Mancini, M. Trevisan, J. Hum. Hypertens. 8 (1994) 677–681. [4] D.S. Freedman, D.F. Williamson, E.W. Gunter, T. Byers, Am. J. Epidemiol. 141 (1995) 637–644. [5] D. Khanna, J.D. Fitzgerald, P.P. Khanna, S. Bae, M. Singh, T. Neogi, M.H. Pillinger, J. Merill, S. Lee, S. Prakash, M. Kaldas, M. Gogia, F. Perez-Ruiz, W. Taylor, F. Lioté, H. Choi, J.A. Singh, N. Dalbeth, S. Kaplan, V. Niyyar, D. Jones, S.A. Yarows, B. Roessler, G. Kerr, C. King, G. Levy, D.E. Furst, N.L. Edwards, B. Mandell, H.R. Schumacher, M. Robbins, N. Wenger, R. Terkeltaub, Arthritis Care Res. (Hoboken) 64 (10) (2012) 1431–1446. [6] A.M. Hung, R.M. Hakim, Am. J. Kidney Dis. 66 (2015) 125–132. [7] X. Yang, Y. Yuan, C.G. Zhan, F. Liao, Drug Dev. Res. 73 (2012) 66–72. [8] E. Nemati, A. Khosravi, B. Einollahi, M. Meshkati, M. Taghipour, S. Abbaszadeh, J. Renal Inj. Prev. 6 (2) (2017) 142–147. [9] I. Perçin, G. Baydemir, B. Ergün, A. Denizli, Artif. Cells Nanomed. Biotechnol. 41 (2013) 172–177. [10] E. Özgür, N. Bereli, D. Türkmen, S. Ünal, A. Denizli, Mater. Sci. Eng. C 31 (2011) 915–920. [11] A.P. Leshchinskaya, N.M. Ezhova, O.A. Pisarev, React. Funct. Polym. 102 (2016) 101–109. [12] V.A. Davankov, L.A. Pavlova, M.P. Tsyurupa, D.R. Tur, J. Chromatogr. A 689 (1) (1997) 117–122. [13] V. Wernert, O. Schäf, H. Ghobarkar, R. Denoyel, Microporous Mesoporous Mater. 83 (1–3) (2005) 101–113. [14] C.J. Liu, X.Y. Liang, X.J. Liu, Q. Wang, L. Zhan, R. Zhang, W.M. Qiao, L.C. Ling, Appl. Surf. Sci. 254 (21) (2008) 6701–6705. [15] H.Q. Zhang, L. Ye, K. Mosbach, J. Mol. Recognit. 19 (4) (2006) 248–259. [16] G. Baydemir, N. Bereli, M. Andac, R. Say, I.Y. Galaev, A. Denizli, Colloid. Surf. B 68 (2009) 33–38. [17] S. Hajizadeh, C. Xu, H. Kirsebom, L. Ye, B. Mattiasson, J. Chromatogr. A 1274 (2013) 6–12. [18] C. Cristallini, G. Ciardelli, N. Barbani, P. Giusti, Macromol. Biosci. 4 (1) (2004) 31–38. [19] D. Silvestri, N. Barbani, C. Cristallini, P. Giusti, G. Ciardelli, J. Membr. Sci. 282 (1–2) (2006) 284–295. [20] B. Gao, S. Liu, Y. Li, J. Chromatogr. A 1217 (15) (2010) 2226–2236. [21] A.P. Leshchinskaya, I.V. Polyakova, A.R. Groshikova, O.A. Pisarev, E.F. Panarin, Mol. Impr. 1 (2013) 17–26. [22] A.P. Leshchinskaya, N.M. Ezhova, O.A. Pisarev, Russ. J. Appl. Chem. 88 (5) (2015) 820–825. [23] B. Ergün, G. Baydemir, M. Andaç, H. Yavuz, A. Denizli, J. Appl. Polym. Sci. 125 (1) (2012) 254–262. [24] Y. Zheng, V.M. Gunko, C.A. Howell, S.R. Sandeman, G.J. Phillips, O.P. Kozynchenko, S.R. Tennison, A.E. Ivanov, S.V. Mikhalovsky, ACS Appl. Mater. Interfaces 4 (2012) 5936–5944. [25] Y. Zheng, V.M. Gun’ko, C.A. Howell, S.R. Sandeman, G.J. Phillips, O.P. Kozynchenko, S.R. Tennison, A.E. Ivanov, S.V. Mikhalovsky, ACS Appl. Mater. Interfaces 4 (2012) 5936–5944. [26] A. Göçenoğlu Sarıkaya, B. Osman, T. Çam, A. Denizli, 251 (2017) 763–772. [27] I. Langmuir, J. Am. Chem. Soc. 38 (1916) 2221–2295. [28] H.M.F. Freundlich, J. Phys. Chem. 57 (1906) 385–471. [29] E. Tümay Özer, B. Osman, A. Kara, E. Demirbel, N. Beşirli, Ş. Güçer, Environ. Technol. 36 (13) (2015) 1698–1706. [30] S. Lagergren, Handlingar 25 (1898) 1–39. [31] Y.S. Ho, J. Hazard. Mater. 136 (3) (2006) 681–689. [32] W. Plazinski, W. Rudzinski, A. Plazinska, Adv. Colloid. Interface. Sci. 152 (2009) 2–13. [33] T.M.S. Chang, Can. J. Physiol. Pharmacol. 52 (2) (1974) 275–285. [34] P.R. Craddock, J. Fehr, K.L. Brigham, R. Kronenberg, H.S. Jacobs, N. Engl. J. Med. 296 (1977) 769–774. [35] H. Yatzidis, Proc. Eur. Dial. Transplan. Assoc. 1 (1964) 83. [36] G. Pott, B. Voss, J. Lohmann, P. Zundorf, J. Clin. Chem. Clin. Biochem. 20 (1982) 333–335. [37] J.F. Winchester, J.G. Rateliffe, E. Carlyle, A.C. Kennedy, Kidney Int. 14 (1978) 74–81. [38] R.P. Franke, F. Jung, Ser. Biomech. 27 (1–2) (2012) 51–58. [39] S. Braune, M. Lange, K. Richau, K. Lützow, T. Weigel, F. Jung, A. Lendlein, Clin. Hemorheol. Microcirc. 46 (2010) 239–250.
4. Conclusion In this study, a composite cryogel was prepared to act as a supporting matrix for the UA selective nanoparticles. The main purpose was to obtain appropriate blood flow besides selective UA removal. UA adsorption studies in aqueous solution showed that the p(AAm-MMA)MIP cryogel has a high UA adsorption capacity (687.6 mg UA /g at 25 °C, pH 6.0) and the adsorption process was endothermic in nature. The kinetic studies demonstrated that the UA adsorption onto the composite cryogel was sufficiently fast. The cryogel can be used repeatedly without a significant change in UA adsorption capacity. The p (AAm-MMA)-MIP cryogel enabled the removal of UA from human serum with a removal percent of 34.8%. The cryogel treatment caused statistically significant decreases in WBC, RBC and PLT counts. On the other hand, the cryogel treatment did not affect the PT, APTT and INR values. The removal percentage of UA was higher than (34.8%) those of other blood components (not greater than 12.7%) indicating the selectivity of the p(AAm-MMA)-MIP cryogel. Thromboelastography studies clearly showed that the cryogel treatment did not affect coagulation factors in contrast to clot strength resulting from platelet decrease. The statisticaly significant changes in aggregation profiles of platelets also demonstrated the effect of platelet activation resulting from surface contact. As a result, the nanoparticle- embedded p(AAm-MMA)-MIP cryogel system provided valuable informations regarding the composite cryogel system in selective extracorporeal toxin removal and facility for blood cell passage.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Molecularly imprinted composite cryogel for extracorporeal removal of uric acid
T
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Bilgen Osmana, , Engin Sagdilekb, Merve Gümrükçüa, Aslı Göçenoğlu Sarıkayaa a b
Bursa Uludag University, Department of Chemistry, Bursa, Turkey Bursa Uludag University, Department of Biophysics, Bursa, Turkey
A R T I C LE I N FO
A B S T R A C T
Keywords: Uric acid Extracorporeal treatment Cryogel Haemocompatibility Removal
In this study, uric acid (UA)-imprinted poly (hydroxyethyl methacrylate-N-methacryloyl-amido-L-cysteine methyl ester)-Fe3+ [poly(HEMA-MAC)-Fe3+] nanoparticle-embedded poly(acrylamide-methyl methacrylate) cryogel [p(AAm-MMA)-MIP] was synthesized for selective UA adsorption. The nanoparticles were prepared via molecular imprinting. The prepared p(AAm-MMA)-MIP cryogel was characterized by Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and swelling test. The swelling degree of the p(AAmMMA)-MIP cryogel was determined as to 7.56 g H2O/g cryogel. The prepared cryogel was used for UA adsorption from aqueous solution.The effects of pH (4.0–8.0), initial UA concentration (5–40 mg/L), temperature (4 °C, 25 °C and 35 °C) and contact time on the UA adsorption capacity were detailedly investigated. UA adsorption data were applied to Langmuir and Freundlich isotherm models. The adsorption data were well fitted with pseudo-second order kinetic model. The thermodynamic parameters (ΔG ͦ , ΔH ͦ , ΔSo) demonstrated that the adsorption process was endothermic and spotaneous at 4 °C, 25 °C and 35 °C. The cryogel was also used for UA adsorption from human serum. The effects of the composite cryogel treatment on blood cells and hemostatic parameters were evaluated by using hemogram analyses, coagulation studies, thromboelastography and platelet aggregation studies. The results showed that the cryogel treatment has an allowable effect on blood cell counts and hemostatic parameters demonstrating the applicability of prepared composite cryogel for UA removal from human serum.
1. Introduction Uremic syndrome is the terminal clinical manifestation of kidney failure [1]. The metabolic compounds refered to uremic toxins are retained in the body during uremic sendrom and adversely affect the biological functions. Uric acid (UA), a water-soluble uremic toxin, is the end product of human purine-nucleotide metabolism. The physiological concentration of UA in blood plasma varies with age and gender, ranging from 2.0 mg/dL to 6.8 mg/dL [2]. UA levels above these limits cause hyperuricemia. Hyperuricemia is associated with numerous diseases, such as Lesch-Nyan syndrome, gout, cardiovascular diseases and kidney diseases [3,4]. Therefore, decreasing serum UA levels has been proposed as a priority in treating hyperuricemia [5]. The level of uremic toxins generally decreases via haemodialysis (HD) or by using therapeutic enzymes that metabolize these harmful biological compounds [6,7]. However, the UA levels have been reported to not sufficiently decrease in uremic patients even with adequate dialysis. Moreover, increasing the adequacy or duration of dialysis is not
⁎
effective [8]. Therapeutic enzymes can be used intravenously to decrease UA levels, but immunity reduces their half-life in circulation when they are used repeatedly for a long time. Hemoperfusion is an extracorporeal treatment that removes endogenous and exogenous toxic substances from patients’ blood. It has been mainly used for poisonings, hepatic failures and treating uraemia and its complications. Affinity adsorption is a proper alternative to conventional hemoperfusion for removing any undesired substances from the patient’s serum in different pathologies. [9,10]. However, developing affinity sorbents for blood treatment is one of the most important problems because these adsorbents should be highly selective and have a relatively high adsorption capacity for the target toxin. Moreover, they should be reusable, biocompatible and mechanically resistant [11]. Many modified polymeric resins meet these requirements for UA removal [12–14]. However, the adsorption capacities of the resins are low, and, more importantly, they have no selectivity for UA. Molecularly imprinted polymers (MIPs) have a high selectivity for
Corresponding author. E-mail address: [email protected] (B. Osman).
https://doi.org/10.1016/j.colsurfb.2019.110456 Received 3 June 2019; Received in revised form 11 August 2019; Accepted 22 August 2019 Available online 23 August 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
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using the procedure developed in our previous study [26]. A detailed experimental procedure for nanoparticle synthesis are given in the Supporting Information. The following procedure was utilized for synthesis of UA-imprinted PHEMA-MAC-Fe3+ nanoparticle-embedded cryogel [p(AAm-MMA)-MIP]: 0.285 g of N,N′ methylenebis(acrylamide) was dissolved in 10 mL of deionized water. Then, 0.71 g of acrylamide (AAm) and 1.07 mL of methyl methacrylate (MMA) were mixed with an aqueous solution of the UA-imprinted nanoparticles. The prepared solutions were mixed together, and then APS and TEMED were added to the mixture at 0 °C. The mixture was poured into a cold syringe prior to polymerization at −18 °C for 18 h. The p(AAm-MMA)MIP cryogel was excessively washed with deionized water after the cryogel thawed to room temperature. The non-imprinted PHEMA-MACFe3+ nanoparticle-embedded cryogel [p(AAm-MMA)-NIP] was prepared via same procedure with the addition of the non-imprinted PHEMA-MAC-Fe3+ nanoparticles prepared without adding UA. The amount of PHEMA-MAC-Fe3+ nanoparticles (MIP or NIP) in the cryogel was 0.57 mg. FTIR analyses were conducted by Spectrum 100 model FTIR spectrometer (Perkin Elmer, USA). The physical morphology of the cryogels coated with thin layer of gold (100 Å) was monitored via a scanning electron microscope (Carl Zeiss Evo 40, Germany). The swelling degree of the p(AAm-MMA)-MIP cryogel was determined in deionized water. The cryogel was incubated in 200 mL of deionized water at room temperature for 2 h. The cryogel sample was taken from the water and carefully weighed after wiping with filter paper. After that, the cryogel was dried at 40 ͦ C for 24 h and weighed. The swelling degree was calculated using Eq. (1);
their target molecules [15] and have been effectively used as affinity adsorbents to remove toxic compounds from blood [16,17]. MIPs with UA recognition have also been reported in recent years. Cristallini et al., 2004 [18] and Silvestri et al., 2006 [19] prepared acrylic acid‐acrylonitrile copolymer membranes to selectively remove UA from the blood. Functional polyamidoxime/crosslinked polyvinyl alcohol (PAO/ CPVA) microspheres with excellent binding affinity for UA were prepared by Gao et al., 2010 [20]. The polymers based on ethylene glycol dimethacrylate (EGDMA) and dimethylaminoethyl methacrylate (DMAEMA) were also synthesized for selective UA adsorption [21,22]. Today, the use of cryogels that present relatively low flow resistance due to their interconnected macroporous structures has attracted attention in biomedical applications. They have a high mechanical strength and a resistance to chemicals. Composite cryogels, including MIP particles, have also been used for affinity adsorption of toxic compounds from blood. The ion-imprinted beads embedded poly (hydroxyethyl methacrylate) (PHEMA) cryogel was successful for the in vitro removal of iron (III) ions from beta-thalassemic human plasma [23]. Baydemir et al., 2009 [16] prepared a supermacroporous PHEMA cryogel embedded with bilirubin-imprinted particles. It is possible to obtain both low back pressure and selectivity by embedding MIPs in the cryogels, which facilitates separation processes. Zheng et al, 2012 [24] prepared poly (vinyl alcohol) cryogels embedded with activated carbon microparticles to investigate the potential use of cryogelation technology as an activated carbon-anchoring backbone in new hemoperfusion-based applications. Although the targeted uremic toxins were removed effectively, the haemocompatibility of the composite cryogel was very low due to significant decreases in red blood cell (RBC) counts (80%), white blood cell (WBC) counts (> 50%) and platelet (PLT) counts (> 50%). [25]. In this study, a composite cryogel including selective nanoparticles was prepared for UA removal for the first time. The haemocompatibility of the composite cryogel was tested by hemogram analyses, coagulation studies, thromboelastography and platelet aggregation studies. UAimprinted poly (hydroxyethyl methacrylate-N-methacryloyl-amido-Lcysteine methyl ester)-Fe3+ [PHEMA-MAC-Fe3+] nanoparticles were prepared via molecular imprinting. The UA-imprinted PHEMA-MACFe3+ nanoparticles were embedded into poly(acrylamide-methyl methacrylate) [p(AAm-MMA)] cryogel to prepare the composite cryogel [p (AAm-MMA)-MIP]. Adsorption studies were conducted to evaluate the effects of pH, initial UA concentration, temperature and contact time on the adsorbed amount of UA onto the p(AAm-MMA)-MIP cryogel. The adsorption data were used for isothermal, kinetic and thermodynamic analyses. The UA removal performance of the p(AAm-MMA)-MIP cryogel from human plasma was also evaluated together with other blood components.
Swelling degree =
ws − w0 w0
(1)
where ws and w0 were the weight of swelled and dry cryogels, respectively. Macroporosity of the synthesized cryogel was calculated according to Eq. (2). In the procedure, cryogels were first swelled up to equilibrium and weighed (ws ); then squeezed by hand and weighed (wsq ); and finally dried until the constant weight (wd ) obtained. Macroporosity was calculated through following equation:
Macroporosity % =
(ws ) − (wsq ) wd
x100
(2)
2.3. Adsorption experiments UA adsorption experiments were conducted on the basis of continious analysis. UA solutions were circulated through the p(AAmMMA)-MIP cryogel column via a peristaltic pump (Multi channel cassette pumps–205S/CA, Watson Marlow). The cryogel column (bed height: 3 cm, column diameter: 1.4 cm) was allowed to equilibrate with the UA solutions for 8 h at a flow rate of 0.5 mL/min. Then, the absorbances of UA solutions were measured at 286 nm wavelength by UV–vis spectrophotometer (UV-1700, Shimadzu). Eq. (3) was used to calculate the adsorbed amount of UA;
2. Experimental 2.1. Materials Uric acid (C5H4N4O3), ammonium persulfate (APS), methacryloyl chloride and L-cysteine methyl ester hydrochloride were purchased from Sigma (St. Louis, USA). Acrylamide (AAm), hydroxyethyl methacrylate (HEMA) and ethylene glycol dimethacrylate (EGDMA) were purchased from Fluka. N,N,N′,N′-tetramethylethylenediamine (TEMED) and N,N′-methylenebis(acrylamide) (MBAAm) were supplied from Merck. Disposable cups and pins (Clear) and 0.2 M CaCl2 were purchased from Haemonetics Corporation. Adenosine diphosphate (ADP), collagen, and epinephrine were purchased from Bio/Data Corporation (PAR/PAK®II). Elga Flex3 water purification system (Veloia Water Solutions & Technologies, France) was used to prepare purified water.
Q=
(C0 − C ) V m
(3)
where Q is the amount of adsorbed UA (mg/g); Co and C are the initial and final concentrations of UA in aqueous solution, respectively (mg/ L). m is the mass of the nanoparticles used (g) and V is the volume of the aqueous phase (L). To determine the influence of the aqueous phase pH on UA adsorption capacity of the p(AAm-MMA)-MIP cryogel, the initial pHs of the UA solutions were varied in the range of 4.0-8.0. The initial concentration of UA was 5 mg/L. The effect of initial UA concentration was investigated in the concentration range of 5–40 mg/L at pH 6.0. The kinetics of UA adsorption were analysed for 480 min (pH 6.0, initial UA
2.2. Synthesis and characterization of p(AAm-MMA)-MIP cryogel UA-imprinted PHEMA-MAC-Fe3+ nanoparticles were prepared by 2
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concentration: 30 mg/L) at 4 °C, 25 °C and 35 °C.
2.6. Adsorption studies in human serum
2.4. Selectivity and reusability studies
The p(AAm-MMA)-MIP and p(AAm-MMA)-NIP cryogels were used for UA adsorption from human plasma. A blood sample was collected into a tube that included gel. Then, the sample was centrifugated at 5000 g for 15 min to remove blood cells. An undiluted sample and diluted samples (with ratios of 1:2 and 1:8) spiked with 35 mg/L of UA were prepared, for a total volume of 7 mL.The prepared samples were separately passed from p(AAm-MMA)-MIP and p(AAm-MMA)-NIP cryogels for 4 h (flow rate:0.5 mL/min). The removal of blood components (uric acid, urea, creatinine, glucose, total cholesterol, triglyceride, total protein, albumin, Na+, K+, Cl−, Ca2+) was also investigated by interacting 4 mL of the undiluted serum sample with the p(AAm-MMA)-MIP, p(AAm-MMA)-NIP and p (AAm-MMA) cryogels. The initial UA concentration of the blood sample was 85 mg/L. The concentrations of blood components in the treated and untreated serum samples were determined with ARCHITECT® c 16000TM (Abbott Laboratories, Diagnostic Division, Abbott Park, IL, USA). The experiments were repeated three times. The removal percentage was calculated according to Eq. (4).
The experimental procedures for selectivity and reusability studies of the p(AAm-MMA)-MIP cryogel are given in the Supporting Information. 2.5. Blood compatibility studies Procedure of blood collection and statistical analysis methods used for data evaluation are given in the Supporting Information. To avoid platelet activation and artificial hemolysis a syringe (Hamilton) rather than a peristaltic pump was used for a single-pass blood perfusion experiment through the cryogel in blood compatibility studies. 2.5.1. Hemogram analysis Blood samples (4 mL) taken from seven healthy volunteers were collected into tubes that contained EDTA. Firstly, the p(AAm-MMA)MIP cryogel was swollen by saline solution (% 0.9 NaCl, 5 mL). Then, 2 mL of the blood sample was passed from the p(AAm-MMA)-MIP cryogel. The remaining 2 mL of the sample was kept at room temperature as an untreated (control) sample. Hemogram analyses of the samples were performed by CELL-DYN®RubyTM hemogram device (Abbott Laboratories, Diagnostic Division, Abbott Park, IL, USA). The hemogram parameters used for evaluation are given in the Supporting Information.
Removal % =
(CO − C ) X 100 CO
(4)
where CO and C are the initial and final concentrations of serum components before and after the cryogel treatment, respectively. 3. Results and discussion 3.1. Properties of p(AAm-MMA)-MIP composite cryogel
2.5.2. Coagulation tests Blood samples (4 mL) were collected from the seven healthy volunteers whose bloods were also used in the hemogram analyses. The blood samples were collected into tubes that included sodium sitrate (1:9 v/v). After that, 2 mL of the blood sample was passed from the p (AAm-MMA)-MIP cryogel swollen by saline solution (% 0.9 NaCl, 5 mL). The remaining 2 mL of the sample was kept at room temperature as an untreated (control) sample. The prothrombin time (PT), activated partial thromboplastin time (APTT) and the international normalized ratio (INR) for treated and untreated blood samples were automatically generated with BCS® XP System (Marburg, Germany).
The p(AAm-MMA)-MIP cryogel was synthesized by polymerization of AAm, methyl MMA and MBAAm in the presence of UA-imprinted PHEMA-MAC-Fe3+ nanoparticles. Firstly, the composite cryogel was characterized by FTIR analysis. In the FTIR spectrum (Fig. SI1), a broad absorption band arising from NeH stretching was observed around 3300 cm−1. The strong adsorption bands observed at 1655 cm−1 and 1723 cm−1 belong to stretching vibration of amide carbonyl group and ester carbonyl group, respectively. The results showed that the applied polymerization procedure enabled the synthesis of the p(AAm-MMA)MIP cryogel. The swelling degree and macroporosity of the composite cryogel were determined as 7.56 g H2O/g cryogel and 68.27%, respectively. The main purpose of embedding the UA-imprinted PHEMA-MACFe3+ nanoparticles into the cryogel is to take advantage of the cryogel’s superior flow characteristics. The physical morphologies of the p(AAmMMA), the p(AAm-MMA)-MIP and the p(AAm-MMA)-NIP cryogels were monitored by SEM analyses. The SEM photographs with different magnifications are shown in Fig. 1. The p(AAm-MMA) non-embedded cryogel has interconnected macropores (Fig. 1a and b). The surfaces of the macropores were also completely covered with PHEMA-MAC-Fe3+ nanoparticles (Fig. 1c–e). SEM analyses showed that the applied polymerization procedure enabled the preparation of a UA-selective p(AAm-MMA)-MIP composite cryogel. Thus, the interconnected macropores make it possible to take advantage of the cryogel’s relatively low flow resistance.
2.5.3. Thromboelastography Thromboelastogram (TEG) measurements were performed with TEG®5000 Thrombelastograph® Hemostasis System (HAEMONETICS ORPORATION, MA, USA). Blood samples were put into vacutainer tubes that included 0.9 mL 0.106 M sodium citrate as an anticoagulant (citrate-blood ratio, 1:9 v/v). The volume of blood sample (3 tubes) collected from each volunteer was 27 mL. 4 mL of the blood sample was used for TEG measurements. The remaining 23 mL of the sample was kept for aggregation studies. A detailed procedure of TEG measurements is given in the Supporting Information. 2.5.4. Aggregation measurements An optical aggregometer (Platelet Aggregation Profiler PAP-4CD®, Bio/Data Corporation, Montgomery, PA, USA) was used to perform aggregation measurements. The blood samples (23 mL) anticoagulated with sodium citrate (citrate-blood ratio, 1:9 vol/vol) were centrifugated for 10 min at 250gto prepare platelet-rich plasma (PRP). The plateletpoor plasma (PPP) was prepared by centrifugation of the remaining blood at 2000gfor 15 min. The obtained PRP (5 mL) were divided into two parts. The 2.5 mL of the PRP sample was passed from the p(AAmMMA)-MIP cryogel. The remaining 2.5 mL of the PRP sample was kept under room temperature as an untreated (control) sample. The treated and untreated PRP samples were simultaneously studied in the two channels of the aggregometer. A detailed procedure of aggregation measurements is given in the Supporting Information.
3.2. Adsorption studies 3.2.1. pH effect pH is an effective parameter because UA is adsorbed onto the embedded UA-imprinted PHEMA-MAC-Fe3+ nanoparticles via metal-chelate interaction of MAC, Fe3+ and UA. The potentiometric titration studies in our previous study [26] showed that MAC-Fe3+-UA complex forms at pH 4.0 and is stable at pH values lower than 8.0 (Fig SI2). In the pH range of 4.0–8.0, UA reversibly interacts with the MAC-Fe3+ complex depending on the amount of H+ ions in the medium, i.e pH. 3
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Fig. 1. SEM photographs of the p(AAm-MMA) cryogel (a and b); the p(AAm-MMA)-MIP cryogel (c, d and e); the UA-imprinted PHEMA-MAC-Fe3+ nanoparticles (f) at different magnificaions [(a) 1500×, (b) 3000×, (c) 1520×, (d) 3500×, (e) 500× and (f) 12,000×].
However, MAC-Fe3+ complex distrupts due to the protonation of sulphydryl group (-SH) of the MAC monomer at the pH values lower than 4.0. Additionally, the MAC-Fe3+-UA complex hydrolyzes at the pH values higher than 8.0. Therefore, the effect of pH on the adsorbed amount of UA was investigated in the pH range of 4.0–8.0 (Fig. 2). The adsorbed amount of UA onto the p(AAM-MMA)-MIP cryogel did not significantly change in the studied pH range since the pH of the medium is favorable for MAC-Fe3+−UA complex formation. Thus, pH 6.0 was
determined as an optimal pH for UA adsorption studies.
3.2.2. Isothermal studies The influence of initial UA concentration on UA adsorption was investigated at 4 °C, 25 °C and 35 °C. The concentrations of the UA solutions were varied from 5 to 40 mg / L (pH 6.0). The adsorption experiments could not be performed at higher UA concentrations because the maximum solubility of UA in water is 40 mg/L. The results are 4
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Fig. 2. Effect of pH on UA adsorption onto the p(AAm-MMA)-MIP cryogel (Solution volume: 25 mL, initial UA concentration: 5 mg/L, Temperature: 25 ͦ C, Flow rate: 0.5 mL/min, Time: 8 h).
Fig. 4. Effect of temperature on UA adsorption onto the p(AAm-MMA)-MIP cryogel (Solution volume:25 mL, initial UA concentration: 30 mg/L, Temperature: 4 °C, 25 °C, 35 °C, pH 6.0, Flow rate: 0.5 mL/min, Time: 8 h).
adsorbate coverage is limited to one molecular layer. Therefore, the adsorption data can be fitted by both the Langmuir and the Freundlich isotherm models especially at the low solute concentrations [29]. The UA adsorption data were also fitted with both isotherm models in the low UA concentration range (5–40 mg/L). The UA adsorption capacities of the p(AAm-MMA) and the p(AAmMMA)-NIP cryogels were also evaluated in the same concentration range and compared to that of the p(AAm-MMA)-MIP cryogel at 25 °C (pH 6.0) (Fig. SI3). The adsorption capacity of the p(AAm-MMA)-MIP cryogel was greater than that of the p(AAm-MMA)-NIP cryogel. In addition, the p(AAm-MMA) cryogel has no UA adsorption. The superior UA adsorption performance of the p(AAm-MMA)-MIP cryogel demonstrated that the embedded UA-imprinted PHEMA-MAC-Fe3+ nanoparticles have binding sites with a high affinity for UA.
Fig. 3. Effect of initial UA concentration on UA adsorption onto the p(AAmMMA)-MIP cryogel (Solution volume: 25 mL, pH: 6.0, initial UA concentration: 5–40 mg/L, Temperature: 4 °C, 25 ͦ C,35 °C, Flow rate: 0.5 mL/min, Time: 8 h).
3.2.3. Kinetic studies UA adsorption kinetics investigated at 4 °C, 25 °C and 35 °C are depicted in Fig. 4. The initial UA concentration was 30 mg/L at pH 6.0. The equilibrium time was around 180 min, indicating a fast adsorption rate. Additionally, the amount of adsorbed UA increased with an increasing temperature, indicating the adsorption process’s endothermic nature. The results demonstrated that the prepared cryogel was suitable for rapid and selective UA adsorption. The experimental data were used to examine pseudo-first-order kinetic model [30] and pseudo-second-order kinetic model [31] at three temperatures (277, 298 and 308 K). The linear forms of the applied models are given in Table SI2. The parameters of the kinetic models are summarized in Table SI4. The pseudo-second-order model has the highest correlation coefficients (R2), which are all greater than 0.9997. Moreover, q e (calculated) is close to q e (experimental) suggesting the second-order nature of the adsorption process. The results showed that the rate-limiting step may be the chemical adsorption [32].
provided in Fig. 3. The adsorbed amount of UA increased with an increase in the initial UA concentration at three different temperatures. The maximum amount of UA adsorbed onto the cryogel was determined as to 687.6 mg UA / g at 25 °C (pH 6.0). In addition, the adsorbed amount of UA increased from 574.7 mg / g to 777.9 mg / g with an incease in the temperature from 4 °C to 35 °C. The result indicated that the UA adsorption process was endothermic the in nature. The adsorption capacity of the cryogel was also compared to other UA-imprinted adsorbents (Table SI1). The Langmuir and Freundlich isotherm models were applied to the UA adsorption data with respect to initial UA concentrations from 5 mg/L to 40 mg/L at 4 °C, 25 ͦ C and 35 °C The linear forms of the Langmuir isotherm model [27] and the Freundlich isotherm model were given in Table SI2. The Langmuir isotherm model assumes that the adsorption is monolayer, the adsorbent surface is homogeneous and the energy of the binding sites on the adsorbent surface are equal. The Freundlich isotherm model is used to describe adsorption processes of the adsorbent with heterogeneous binding sites. The energies of the binding sites of the adsorbent are not equal and the interaction between adsorbate molecules is also possible [28]. The calculated parameters for Langmuir and Freundlich isotherm models are shown in Table SI3. Both the Freundlich and the Langmuir isotherm models offered high correlation coefficients for all temperatures and well fitted the adsorption data. The Freundlich isotherm equation is an empirical equation and can be derived from the Langmuir equation by assuming that the binding sites are heterogeneous. The Freundlich isotherm model assumes that the adsorbed amount of solute increases with increasing concentration. Conversely, the Langmuir isotherm supposes that the adsorbed amount of solute reaches a constant value at high concentrations since the extent of
3.2.4. Thermodynamic parameters Thermodynamic parameters; Gibbs free energy change ( ΔGo ), entropy change ( ΔS o ) and enthalpy change ( ΔH o ) for UA adsorption onto p(AAm-MMA)-MIP cryogel were calculated using Van’t Hoff equation and Eq. (6) and are discussed in the Supporting Information (Table SI5).
lnK =
ΔS o ΔH o 1 − ( ) R R T
ΔGo = ΔH o − TΔS o
(5) (6)
3.3. Selectivity and reusability of the p(AAm-MMA)-MIP cryogel The p(AAm-MMA)-MIP cryogel has a high selectivity when theophylline was used as a competitor molecule. The results of selectivity 5
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cryogel also depleted insignificant amount of creatinine and glucose probably because these compounds can act as a ligand to coordinate Fe3+ ions with oxygen and nitrogen atoms in the structure. The removal percent of UA was 34.8% for p(AAm-MMA)-MIP cryogel. Consequently, the p(AAm-MMA)-MIP cryogel with a selectivity for UA has a usage potential as a hemoperfusion adsorbent for UA removal from human blood with allowable effects on other plasma components. 3.5. Effects of the p(AAm-MMA) composite cryogel treatment on blood cells and hemostatic parameters Hemoperfusion is an alternative blood purification procedure in which whole blood is exposed directly to sorbent materials with the capacity to selectively or nonselectively bind endogenous or exogenous toxins. After contact with blood, however, the adsorbent entraps red blood cells, platelets and leukocytes. Therefore, the effects of the p (AAm-MMA)-MIP cryogel treatment on blood cells’ counts were rigorously investigated by hemogram analyses, and the significance of differences between samples was statistically evaluated.. The changes in hemogram parameters that resulted from the cryogel treatment are provided in Table SI7. The significant decrease in HTC counts (8.6%, p < 0.01) after the cryogel treatment reflects the total decrease in white blood cell counts, red blood cell counts and platelet counts. The percentage decreases were 6.5%, 8.3% and 10.7% for WBC, RBC and PLT counts, respectively. Significant decreases were also observed in MONO counts (14%, p < 0.01), probably because they are the largest white blood cells, and in NEU counts (8.6%, p < 0.01) because they are highly abundant WBCs. However, thrombocytopenia, leukopenia, anaemia and a decrease in fibrinogen are the commonly reported adverse reactions to hemoperfusion [33]. For instance, transient leukopenia is commonly observed during hemoperfusion in humans and may result from a complement activation of surface contact, with a margination of leukocytes similar to that observed during haemodialysis. [34]. The decrease in RBC counts was statistically significant (8.3%, p < 0.01). The parameters related to red blood cells, such as HGB (8.8%, p < 0.01), MCV (0.33%, p < 0.01) and RDW (2.43% p < 0.01), were also affected by cryogel treatment. Moreover, the decreases in MCV and RDW counts clearly demonstrated that the large erythrocytes were trapped in the cryogel. The PLT count was also affected by the cryogel treatment, presenting a significant decrease (10.7%) despite platelets being the smallest components of blood. The result probably proceeded from the platelets’ activation and subsequent adherence to the cryogel surface. However, profound platelet depletion was commonly observed in hemoperfusion devices and platelet loss in current hemoperfusion devices is about 30% or less [35]. While the decrease in PLT counts was 10.7% for a single-pass blood perfusion experiment, the decrease in PLT counts was 12.4% when the blood sample interacted with the p(AAm-MMA)-MIP cryogel for 3 h (data not shown). Additionally, no significant decreases were detected in the other hemogram parameters, such as EOS, BASO, LYM, MCH, MCHC and MPV. Consequently, the p(AAm-MMA)-MIP cryogel treatment has an allowable effect on blood cells. The adsorption or activation of coagulation factors is a frequent problem during clinical hemoperfusion. The most significant change occurs in the fibronectin and fibrinogen concentration [36], even if the polymer-coated activated charcoal is used as an adsorbent [37]. Therefore, the p(AAm-MMA)-MIP cryogel treatment’s effect on coagulation parameters was evaluated by measuring the prothrombin time (PT), the international normalized ratio (INR) and the activated partial thromboplastin time (APTT). The PT and APTT times and the INR values presented in Table SI8 clearly show that coagulation factors were not affected by the cryogel treatment because there is no statistically significant change in PT or APTT times or INR values (p > 0.05) after the cryogel treatment. A hemostatic test called thromboelastography measures the
Fig. 5. UA adsorption from human serum samples spiked with 35 mg/L UA (Solution volume: 7 mL, initial UA concentration: 35 mg/L, Temperature: 25 ͦ C, Flow rate: 0.5 mL/min, Time: 4 h, n = 3).
studies are detailedly discussed in the Supporting Information (Fig SI4, Table SI6). The p(AAm-MMA)-MIP composite cryogel was also used for ten successive adsorption/desorption cycles (Fig. SI5). UA interacts with the p(AAm-MMA)-MIP cryogel via metal-chelate interaction with Fe3+ ions. Therefore, the adsorbed UA can be desorbed easily by decreasing the pH of the medium. The result showed that the prepared cryogel can be utilized for UA adsorption without a decrease in UA adsorption performance. 3.4. UA adsorption from human serum Human serum is a complex mixture including electrolytes, antigens, antibodies, hormones and any exogenous substances. The p(AAmMMA)-MIP cryogel was used for UA adsorption from human serum to investigate the cryogel’s selectivity in a real sample. The undiluted serum sample and the diluted (with ratios of 1:2 and 1:8) serum samples, which were spiked with 35 mg/L UA, interacted with the p(AAmMMA)-MIP cryogel. The efficiency of the p(AAm-MMA)-NIP cryogel was also evaluated to show the effectiveness of the molecular imprinting. The results are provided in Fig. 5. The adsorbed amounts of UA onto the p(AAm-MMA)-MIP cryogel were 148.6, 157.1 and 186.6 mg/g for the undiluted sample, the diluted sample in the ratio of 1:2 and the diluted sample in the ratio of 1:8, respectively. The amounts of UA adsorbed onto the p(AAm-MMA)-NIP cryogel were lower than those adsorbed onto the p(AAm-MMA)-MIP cryogel for all studied samples. Although all the serum samples were spiked with 35 mg/L UA, the highest amount of UA adsorption was obtained for the sample diluted at the ratio of 1:8, suggesting that low-concentration plasma components were not sufficient to replace UA. However, the adsorbed amounts of UA calculated for the undiluted sample and the diluted sample in the ratio of 1:2 were not significantly different, indicating that the p(AAm-MMA)-MIP cryogel has a high affinity for UA molecules. Additionally, the percent of removed plasma components, such as Ca2+, Cl−, K+, Na+, creatinine, glucose, albumin, cholesterol, total protein, triglyceride and urea, was investigated to assess the effectiveness and possible side effects that the p(AAm-MMA)-MIP cryogel may improve in hemoperfusion. The percent of removed plasma components for the non-embedded p(AAm-MMA), the p(AAm-MMA)-MIP and the p (AAm-MMA)-NIP cryogel treatments are depicted in Fig. SI6. The results showed that the p(AAm-MMA) cryogel has no adsorption. The p (AAm-MMA)-MIP and p(AAm-MMA)-NIP cryogels did not deplete albumin, cholesterol, total protein and triglyceride. The removal percent of urea was 12.3% for p(AAm-MMA)-NIP cryogel while the p(AAmMMA)-MIP cryogel has no urea adsorption owing to the selectivity of the cryogel. The removal percentages of electrolytes were not greater than 12.7% for p(AAm-MMA)-MIP cryogel. The p(AAm-MMA)-MIP 6
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Acknowledgement
dynamics of clot formation and the stability and strength of clots. The thromboelastogram parameters extend our knowledge about the effect of foreign material contact on hemostatic parameters. Therefore, the TEG parameters of the treated and untreated blood samples were determined and summarized in Table SI8. The graph showing the TEG curves is also depicted in Fig SI7. The results indicate that the cryogel treatment caused statistically significant decreases in maximum amplitude (MA, mm, p < 0.001), clot strength (G, dyn/cm2, p < 0.01) and amplitude at 60 min (A60, mm, p < 0.05)—suggesting that cryogel column treatment decreased clot strength and amplitude. The decrease in platelet counts (10.7%) after the cryogel treatment probably caused a decrease in clot strength because MA (mm) and G (dyn/ cm2) are relate directly with the number and function of the platelets. Other TEG parameters such as angle (α, degrees) and reaction time (R, min) relate to coagulation factors and did not change significantly. These results clearly show that the cryogel treatment did not affect the coagulation parameters, which is consistent with the results of coagulation studies. Platelets mainly function to aggregate and form blood clots at injured vessel walls. Platelets’ contact with foreign-material surfaces results in adhesion and subsequent activation, depending upon the properties of the surface (e.g. its wettability, roughness, polarity and chemical composition) [38]. Surface roughness induces platelet adherence and hydrophilic surfaces prevent cell attachment [39]. Therefore, the aggregation profiles of platelets were also investigated to determine the effect of a cryogel treatment. The changes in maximum aggregation (%) and slope parameters obtained by different aggregation agents (e.g. ADP, epinephrine and collagen) are presented in Table SI8. The aggregation curves for ADP, epinephrine and collagen are also shown in Fig SI8. The p(AAm-MMA)-MIP cryogel treatment caused statistically significant increases in maximum aggregation (%) values for all studied stimulants (p < 0.05). However, the percentage increases in maximum aggregation (%) values were different for ADP (16.7%), collagen (5.3%) and epinephrine (7.5%), probably because the agents have distinct intracellular pathways. Thus, the platelets’ aggregation behaviour was affected by the cryogel treatment because the platelets become proactive.
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4. Conclusion In this study, a composite cryogel was prepared to act as a supporting matrix for the UA selective nanoparticles. The main purpose was to obtain appropriate blood flow besides selective UA removal. UA adsorption studies in aqueous solution showed that the p(AAm-MMA)MIP cryogel has a high UA adsorption capacity (687.6 mg UA /g at 25 °C, pH 6.0) and the adsorption process was endothermic in nature. The kinetic studies demonstrated that the UA adsorption onto the composite cryogel was sufficiently fast. The cryogel can be used repeatedly without a significant change in UA adsorption capacity. The p (AAm-MMA)-MIP cryogel enabled the removal of UA from human serum with a removal percent of 34.8%. The cryogel treatment caused statistically significant decreases in WBC, RBC and PLT counts. On the other hand, the cryogel treatment did not affect the PT, APTT and INR values. The removal percentage of UA was higher than (34.8%) those of other blood components (not greater than 12.7%) indicating the selectivity of the p(AAm-MMA)-MIP cryogel. Thromboelastography studies clearly showed that the cryogel treatment did not affect coagulation factors in contrast to clot strength resulting from platelet decrease. The statisticaly significant changes in aggregation profiles of platelets also demonstrated the effect of platelet activation resulting from surface contact. As a result, the nanoparticle- embedded p(AAm-MMA)-MIP cryogel system provided valuable informations regarding the composite cryogel system in selective extracorporeal toxin removal and facility for blood cell passage.
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