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Antifouling zwitterionic hydrogel coating improves hemocompatibility of activated carbon hemoadsorbent
Accepted Manuscript Regular Article Antifouling zwitterionic hydrogel coating improves hemocompatibility of activated carbon hemoadsorbent Nana Cai, Qingsi Li, Jiamin Zhang, Tong Xu, Weiqiang Zhao, Jing Yang, Lei Zhang PII: DOI: Reference:
S0021-9797(17)30417-4 http://dx.doi.org/10.1016/j.jcis.2017.04.024 YJCIS 22236
To appear in:
Journal of Colloid and Interface Science
Received Date: Revised Date: Accepted Date:
24 February 2017 6 April 2017 7 April 2017
Please cite this article as: N. Cai, Q. Li, J. Zhang, T. Xu, W. Zhao, J. Yang, L. Zhang, Antifouling zwitterionic hydrogel coating improves hemocompatibility of activated carbon hemoadsorbent, Journal of Colloid and Interface Science (2017), doi: http://dx.doi.org/10.1016/j.jcis.2017.04.024
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Graphical abstract
Antifouling zwitterionic hydrogel coating improves hemocompatibility of activated carbon hemoadsorbent Nana Cai,a,b,c Qingsi Li,a,b,c Jiamin Zhang,a,b,c Tong Xu,a,b,c Weiqiang Zhao,a,b,c Jing Yanga,b,c and Lei Zhang* a,b,c a Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China b Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin, 300072, PR China c Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin, 300072, PR China Corresponding author at: Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China. E-mail address: [email protected] Cell: 86-13502038015
Abstract Activated carbon has been widely used in hemoperfusion treatments. However, its performance has been significantly compromised by their poor hemocompatibility. In this work, we developed a novel antifouling adsorbent based on zwitterionic poly-carboxybetaine (PCB) hydrogel and powdered activated carbon (PAC) to improve hemocompatibility. We found this new adsorbent (PCB-PAC) was highly stable with negligible leakage of activated carbon debris. It could efficiently resist protein adsorption and avoid any hemolysis effect. The adsorption performance of PCB-PAC for methylene blue was not influenced in a single protein solution or even in 100%
fetal bovine serum (FBS), in which pristine PAC lost 50% of its adsorption ability. The isotherms results showed that the adsorption process of PCB-PAC fitted the Langmuir isotherm well, indicating that the PAC particles were homogenously distributed in the PCB hydrogel matrix. Moreover, PCB-PAC could also adsorb bilirubin molecules bound to albumin in solution, while pristine PAC showed no discernible adsorption effect. Findings in this work hold great potential to significantly improve the performance and efficiency of current extracorporeal devices for removing toxins from blood directly.
Keywords: zwitterionic hydrogel anti-biofouling activated carbon adsorption hemocompatibility
Introduction Basing on adsorption, hemoperfusion (HP) technology can be used to remove toxic substances from the blood of patients, thus it has been widely used clinically in the treatment of severe intoxications, nephritic, hepatic/multiorgan insufficiency, autoimmune diseases, sepsis, etc [1-3]. Unlike hemodialysis (HD) and hemofiltration (HF), which mainly remove small and hydrophilic molecules from blood, a unique ability of hemoperfusion is to efficiently adsorb molecules with larger molecular weight and/or high protein-binding affinity [4-6]. The curative efficiency of hemoperfusion critically depends on the performance and hemocompatibility of the adsorbents [3, 7]. Therefore, the development of high efficient and hemocompatible adsorbents is the main focus in hemoperfusion research and development [8]. Activated carbon is the most commonly used material as a nonspecific adsorbent in a clinical setting due to its strong adsorption ability and low cost [9-11]. However, pristine activated carbon particles present poor blood compatibility which hinders their successful practical applications: firstly, upon contacting with blood, they suffer from non-specific blood protein adsorption which would cover the adsorbents’ surface and significantly reduce their adsorption ability; secondly, activated carbon materials would result in serious clinical side effects such as particulate microemboli formation, hemolysis and blood coagulation [12-14]. To solve this problem, Chang et al. introduced a microencapsulation process that could cover the activated carbon particles with a polymeric coating [15]. In this way, the coating could directly contact with blood instead of bare activated carbon, thus the hemocompatibility and the hemoperfusion performance could be improved [16]. In the pioneering studies of Chang, a number of coating materials such as cellulose nitrate, albumin-collodion, cellulose acetate, and polyamide have been evaluated [17-19]. Later, Andrade et al. investigated the use of hydrogel as coating material for activated carbon particles [20]. Hydrogel is a crosslinked hydrophilic polymeric network with high water content and good biocompatibility [21]. It could ensure better diffusive permeability and provide biomimetic mechanical strengths [22-24]. These work lead to the commercialization of polymer coatings (cellulose acetate, polyhydroxyethylmethacrylate, polyacrylic, etc) based activated carbon adsorbents [13]. However, current coating materials still could not fully
eliminate the negative influence in hemoperfusion applications and therefore the development of novel polymer coatings with better performance is still highly desired [14, 16].
Ideal coating materials for activated carbon must possess excellent blood compatibility without hemolysis, protein adsorption or coagulation, while should also be highly permeable to target toxins [16, 25]. Recently, zwitterionic materials have received increasingly attention in many biomedical applications, attributed to their excellent “antifouling” properties to resist non-specific protein adsorption as well as cell or bacteria adhesion. Owing to the opposing but balanced charges in each moiety, zwitterionic-based materials can strongly bind water molecules via electrostatically induced hydration, which poses an obstacle for protein settlement. Moreover, zwitterionic hydrogels exhibit high water content and large mesh size which could benefit the diffusion of molecules through the hydrogel and provide biomimetic mechanical strengths [23, 26]. Among zwitterionic materials, carboxybetaine (CB) is a very interesting and unique one. The structure of CB monomer is similar to glycine betaine, a natural compound in the human body for osmotic regulation. PCB coated surface could reduce protein adsorption to an undetectable level even in 100% human blood serum [26]. Moreover, PCB possess many unique properties: Zhang et al. in 2008 reported that polyCBMA exhibited unique anticoagulant activity which made it a promising polymer for blood-contacting applications [27]. In 2012, Hsiu-Wen Chien et al. proved that PCB hydrogel was highly compatible with living cells and could maintain the viability of the encapsulated cells. This also proved the excellent permeability of PCB hydrogel to nutrients and cell wastes [28]. In more recent studies, PCB hydrogel was shown to resist the foreign body reaction when implanted in mice and effectively enhance protein stability [29-31]. These findings motivated us to explore the application of PCB hydrogel as the coating material for activated carbon particles. In the present study, we developed a novel adsorbent for hemoperfusion based on PCB hydrogel and PAC particles. We demonstrated that the new adsorbent was highly hemocompatible with fresh blood and its adsorbing ability was not compromised even in 100% blood serum. Furthermore, the adsorption of bilirubin was investigated to show the versatile adsorption abilities of the PCB-PAC. The results showed that PCB-PAC would be a potential biomedical material for detoxification of biological fluids, in particular blood or blood plasma of patients.
2. Materials and methods 2.1. Materials Methylene blue (MB) was purchased from J&K Scientific Ltd. (Beijing, China). Bovine serum albumin (BSA) and bilirubin (BR) were purchased from Solarbio Life Science. Fetal bovine serum (FBS) was obtained from Gibco. Ammonium persulfate (APS) and N,N,N’,N’-Tetramethylethylenediamine (TEMED) were purchased from Across Organics. N,N’-Methylenebis (acrylamide) (MBAA) was purchased from Alfa Aesar. Tetraethylene glycol dimethacrylate (TEGDMA) and Sodium chloride were p-
urchased from Sinopharm Chemical Reagent Bejing Co., Ltd. (Beijing, China). Phosphate buffer saline (PBS) solution (0.01 M, pH 7.4) was made from PBS powder purchased from Solarbio Life Science. Ethanol, ethylene glycol and sodium hydroxide were purchased from Yuanli, Tianjin. Milli-Q water (18.2 MΩ cm-1) was used in all experiments. 2-Hydroxyethyl methacrylate (HEMA) was purchased from Acros Organics. Poly(ethylene glycol) methacrylate (PEGMA Mn 500) was obtained from Sigma-Aldrich. Carboxybetaine methacrylate (CBMA) was synthesized as previously reported [23, 32].
2.2. Adsorbents preparation The granular activated carbon was grinded to microparticles and then sieved to remove particles greater than 50 microns. To prepare PCB-PAC, CBMA monomer and MBAA crosslinker were dissolved in 1 M NaCl at a concentration of 40% (wt/vol) and a crosslinking ratio of 1% (molar percent of monomer) [33]. The solution was homogenized by sonication in an ice bath for 30 min. Then, PAC particles were added at 160 mg per 1 mL solution. After PAC addition, the mixture was stirred overnight and then sonicated for 10 min to obtain a homogenous slurry. After this, 1 mg of the initiators (APS) and 1 μL the promoters (TEMED) were added to the solution to initiate polymerization between glass microscope slides separated by 0.5 mm thick polytetrafluoroethylene (PTFE) spacers at 37 ℃ for 30 min. The sample was then removed from the slides and immersed in PBS for equilibration. The buffer was changed every 12 hours for 5 days. [23, 32, 34] Similarly, to prepare PHEMA-PAC, HEMA monomer and the crosslinker (TEGDMA) were added to a mixed solvent (ethanol/ethylene glycol/H2O =1:1.5:1.5, volume ratio) at a concentration of 40% and a crosslinking ratio of 1% [35]. Then, PAC particles (170 mg/mL) were added and the solution was stirred overnight and sonicated well. Finally, 2 mg of the initiators and 1.5 μL the promoters were added to initiate the polymerization as described above. PEGMA-PAC was prepared by mixing PEGMA monomer and the crosslinker (TEGDMA) to ethylene glycol at a concentration of 40% and a crosslinking density of 1% [36]. Then, PAC particles (167 mg) were added to the solution (1 mL) and the mixture was stirred overnight and sonicated well. Finally, 3 mg of the initiators and 2 μL the promoters were added to the mixed solution to initiate the polymerization as described above. Pure PCB, PHEMA and PEGMA hydrogels without PAC were prepared as control samples. All samples were cut into 1 mm-sized pellets and washed with PBS before use.
2.3. Characterizations 5 mm diameter disks of all hydrogel samples with or without PAC were tested using a WDW-5 electromechanical tester (Beijing Time Technologies CO., LTD, China) with a 200 N load cell at a rate of 0.5 mm/min. The Young’s modulus was calculated from 10% to 20% strain. The equilibrium swelling degree of the samples was performed by calculating the
volume ratio of samples after and before swelling. The diameter and thickness of the fully swollen disks were measured using the vernier caliper. The equilibrium swelling degree (SD) was determined using the following equation:
(2-1) Where Vs and Vr are the volumes of the fully swollen and the unswollen disks; DS and DR are the diameters of the fully swollen and the unswollen disks; Ls and Lr are the thicknesses of fully swollen and unswollen disks, respectively. According to the swelling degree and mechanical properties, mesh size of the hydrogel coatings (distance between crosslinks) can be determined by the following two equations:
(2-2) Where is the stress at a particular strain, in units of Pa, α is the deformation ratio related to the stress determined as the ratio of elastically deformed length L to initial length L0 of a crosslinked hydrogel under compression. R is the universal gas constant, T is absolute temperature, 2 is the volume fraction of polymer at equilibrium (in the fully swollen hydrogel), and o is the volume fraction of polymer in the relaxed state (non-swollen, but not dehydrated hydrogel).
(2-3) where NA is Avagadro’s number. The value of hydrogel coatings. [23]
is definitely mesh size of the
2.4. Stability tests The encapsulation stability of the PAC microparticles in different hydrogels was evaluated by incubating the samples in 1 mL 0.9% NaCl solution containing 300 units of injectable heparin sodium salt according to the previous report [14]. The mixtures were incubated in a shaking water bath at 175 rpm, 25 ℃ for 60 min and then the optical density of the supernatant was measured at 600 nm for the leakage of fine microparticles. This experiment was repeated three times by adding fresh solution each time.
2.5. Adsorption tests To study the adsorption behavior of different hydrogel coated absorbents, PCB-PAC, PEGMA-PAC and PHEMA-PAC samples (all containing 10 mg PAC) were placed into flasks containing 20 mL of 50 mg/L MB in PBS solution. 10 mg pristine PAC was studied as the control sample. The flasks were placed in a shaking water bath of 175 rpm at 25 ℃. The residual concentrations were measured at different time points using ultraviolet-visible (UV-Vis) absorption spectroscopy at a wavelength of 663 nm.
The removal efficiency (%) was determined using the following equation:
(2-4) where Re, C0 and Ct are the removal efficiency of MB by the adsorbents at time t (%), the initial MB concentration (mg/L) and the residual MB concentration at time t (mg/L), respectively.
2.6. Adsorption isotherms The MB adsorption capacity of the pristine PAC and the PCB-PAC was investigated by batch adsorption experiments with MB in PBS solution. 10 mg pristine PAC and PCB-PAC (containing 10 mg PAC) were equilibrated in 20 mL solutions with different MB concentrations in a shaking water bath of 175 rpm at 25 ℃ for 24 h. The amount of adsorbed MB was calculated from the residual amount of MB in the solution via the mass balance. The amount of MB adsorbed at equilibrium qe (mg/g) was calculated according to the following equation:
(2-5) In this equation, qe is the amount of solute adsorbed per unit weight of the PAC particles at equilibrium (mg/g) and Ce is equilibrium concentration of MB in solution after adsorption (mg/L).
2.7. Hemolysis evaluation In this work, the hemolysis effect was evaluated by incubation of the hydrogel coated PAC and pristine PAC in diluted blood containing 20% fresh blood and 80% normal saline at 37 ℃ for 30 min. Blood samples diluted with 80% normal saline and deionized water in the absence of adsorbents were used as negative control and positive control, respectively. Then, the blood samples were centrifuged at 1000 rpm for 10 min and the colors of the supernatants were evaluated. The hemolysis ratio (HR) was determined by measuring the absorbance of supernatants at 541 nm by UV-Vis spectroscopy and calculated with the following equation: (2-6) Where AS, AN and AP are the absorbance of the samples, the negative control and the positive control, respectively. [37]
2.8. Performance in biological fluids To test the adsorption performance of pristine PAC and PCB-PAC in biological fluids, MB was dissolved in PBS solution, PBS solution containing 40 mg/mL BSA and 100% FBS. 10 mg pristine PAC and PCB-PAC (containing 10 mg PAC) were incubated with 20 mL of MB in different solutions with a same initial concentration of 50 mg/L. The samples (0.1 mL) were taken at different time points and were analyzed the
residual concentrations. The removal efficiency at different time points was measured using the equation described above.
2.8. Adsorption of bilirubin from albumin To evaluate the ability of PCB-PAC to isolate molecules bound to plasma proteins, bilirubin-albumin (BR-BSA) solution was prepared by dissolving solid bilirubin in 0.1 M NaOH, then adding the resulting solution into BSA solution, and the final pH was adjusted to 7.4 [38]. PCB-PAC particles (containing 30 mg PAC for each test) were placed into flasks containing 5 mL BR-BSA solution (150 mg/L BR, 40 mg/mL BSA). 30 mg pristine PAC was used as the control sample. The flasks were placed in a shaking water bath of 175 rpm at 25 ℃. The whole procedure was conducted in the dark to avoid photodegradation of BR. The residual concentrations were measured at different time points using ultraviolet-visible (UV-Vis) absorption spectroscopy at a wavelength of 460 nm. The equilibrium adsorption amount of BR qe (mg/g) was measured using the equation described above.
3. Results and Discussion 3.1. Physical properties of adsorbents The physical properties of the samples are summarized in Table 1. The compressive modulus and the break strain of the hydrogel coated PAC samples were slightly higher than those of the corresponding pure hydrogels. The PCB-PAC possessed larger break strain (71%) than PEGMA-PAC (31%), indicating PCB-PAC could better maintain their integrity during the adsorption process. In contrast, PEGMA-PAC displayed a higher compressive modulus but a lower break strain. The hydrogel coated PAC samples exhibited the similar swelling degree as that of the pure hydrogels. Due to the strong hydration properties, PCB hydrogel coating possessed the largest swelling degree (1.69), while that of PEGMA hydrogel coating was 1.12. In comparison, PHEMA hydrogel coating showed much lower swelling degree (0.79), which meant it slightly shrank in aqueous solutions. Larger mesh size allowed better mass transfer for molecules through the hydrogel coating which was crucial in hemoperfusion applications. For PEGMA and PHEMA hydrogel coatings, the mesh size distributions were narrow and the diameters were 2.26 nm and 1.94 nm, respectively. Attributed to the largest swelling degree, the mesh size of PCB hydrogel coating (5.59 nm) was twice more than those of PEGMA and PHEMA hydrogel coatings which ensures higher diffusive permeability.
3.2. Adsorbents stability One important advantage of the hydrogel encapsulation method is to avoid the leakage of PAC debris, which may cause severe side effects clinically. The stability tests (Fig. 1) showed that PCB-PAC was highly stable with negligible release of fine
PAC microparticles thanks to its high break strain and good stability. In comparison, PEGMA-PAC and PHEMA-PAC were much less stable and showed obvious PAC microparticles leakage. Due to the poor mechanical strength, PEGMA-PAC tended to collapse under shaking, while although PHEMA hydrogel was much stronger, the encapsulation appeared unstable possibly due to the poor compatibility between PHEMA hydrogel and the PAC particles.
3.3. Adsorption properties MB (molecular weight 319.86 Da) was chosen as a model molecule. It has been commonly used to evaluate the adsorption performance of activated carbon in aqueous solutions and study the effects of coating materials on adsorption behaviors [2, 39, 40]. All three types of hydrogel coated PAC samples were prepared as 1 mm-sized particles. The surface area of hydrogel particles (containing 10 mg PAC in each test) was about 10-4 m2, while the surface area of 10 mg PAC was about 12 m2, which was much larger and dominated the adsorption capacity. The different adsorption behaviors of the samples are shown in Fig. 2. It could be seen that all three types of hydrogel coated PAC samples showed a decreased adsorption profile compared with pristine PAC. This was because the hydrogel coatings performed as mass transfer barriers for MB. However, thanks to the large mesh size of PCB hydrogel coating, the adsorption performance of PCB-PAC was not significantly influenced. In comparison, mesh sizes of PHEMA and PEGMA hydrogels were much smaller, thus their adsorption rates were delayed and their adsorption capacities were significantly decreased. The PCB-PAC removed almost 100% of MB after 7.5 h, similar to pristine PAC, while the removal ratios of PEGMA-PAC and PHEMA-PAC were only 52% and 31%, respectively. This was possibly caused by a large ratio of adsorption sites in PAC were made inaccessible by the compact structure of PEGMA and PHEMA hydrogels. Therefore, PCB-PAC was found to be the most suitable adsorbent among the three samples tested. To further understand the adsorption process of PCB-PAC, we investigated the adsorption kinetics and isotherms in PBS solution. Two widely used kinetic models, pseudo-first-order model (Eq. 3-1) and pseudo-second-order model (Eq. 3-2), were used to study the adsorption kinetic behaviors of MB on pristine PAC and PCB-PAC [41, 42]. (3-1)
(3-2) In Eq. (3-1), qt is the amount of MB adsorbed per unit weight of PAC particles at time t (mg/g); k1 is the rate constant of pseudo-first-order model (min-1). In Eq. (3-2), k2 is the rate constant of pseudo-second-order model (g mg-1 min-1); h=k2qe2 is the initial sorption rate (mg g-1 min-1). The fitting results using pseudo-first-order model and pseudo-second-order model are shown in Fig. 3. The rate constants and the correlation coefficients related to each
model were calculated and listed in Table 2. All the experimental data showed better compliance with pseudo-second-order model in terms of higher correlation coefficient values (R2>0.998). The experimental qe values (qe.exp) showed a very good agreement with the theoretical ones (qe.cal), calculated by pseudo-second-order model. The initial adsorption rate (h= k2qe2) obtained from this model validated the experimental removal results that the pristine PAC had a faster initial adsorption rate than the PCB-PAC under similar conditions. This model was valid for pristine PAC and PCB-PAC which could be utilized to describe the adsorption kinetics and to forecast the adsorption capacity at t (time). The intraparticle diffusion model (Eq. 3-3) was used to elucidate the diffusion mechanism during the adsorption process [43]. (3-3) In Eq. (3-3), K3 and C are the rate constant of the intraparticle diffusion model (mg g-1 min1/2) and the intercept, respectively. The value of C gives an idea about the thickness of the boundary layer. A larger value of C represents a greater boundary layer diffusion effect. If the value of C is 0, the intraparticle diffusion is the sole rate controlling step. If the value of C is not 0, the intraparticle diffusion is not the only rate controlling step and other factors may also control the process of adsorption. As shown in Fig. 4, the removal of MB was found to be rapid at the initial period which was controlled by the external surface diffusion of adsorbate or the boundary layer diffusion. The adsorption behaviors were followed by a gradual adsorption stage where intraparticle diffusion was rate limiting. The final stage was achieved equilibrium of which the intraparticle diffusion started to slow down due to the extremely low MB concentration left in the solution. [43] The corresponding parameters are calculated and summarized in Table 2. It was found that the intraparticle diffusion was not the only rate controlling step in the adsorption process of pristine PAC and PCB-PAC (C≠0). The pristine PAC showed an initial fast adsorption stage and then reached equilibrium. These results indicated that a significant boundary layer diffusion effect occurred in the adsorption process of pristine PAC. It was consistent with the results in literatures which had shown that the boundary layer diffusion was primary rate controlling step in small size adsorbents [43]. Whereas in the adsorption process of PCB-PAC, the diffusion of molecules through the polymeric matrix into the PAC particles was the primary rate controlling step. This adsorption behavior was well correlated with the structure of the PCB-PAC.
3.4. Adsorption Isotherms The correlation of the equilibrium adsorption capacity and equilibrium concentration can be represented by the adsorption isotherms. Two models are commonly used for studies of adsorption, the Langmuir isotherm (Eq. 3-4) and the Freundlich isotherm (Eq. 3-6). This experiment through the modeling analysis provided an insight into the adsorption mechanism and surface properties of adsorbents. The Langmuir isotherm is suitable in homogeneous systems in which all adsorbing sites possess equal affinity for the adsorbate, while the Freundlich model refers to multilayer adsorption indicati-
ng non-uniform distribution of binding affinities over the heterogeneous systems. The essential characteristics of the Langmuir isotherm can be described by a separation factor RL (Eq. 3-5). Non-Linear form:
Non-Linear form:
Linear form:
(3-4)
（
(3-5)
Linear form:
(3-6)
In Eq. (3-4), qm is the maximum adsorption capacity of adsorbent (mg/g); KL is the Langmuir constant associated to the affinity of binding sites. In Eq. (3-5), RL is the separation factor indicating that the adsorption process is favorable or not. According to this model, when RL is between 0 and 1, the adsorption system is favorable. In Eq. (3-6), KF (mg/g)(L/mg)1/n is the Freundlich constant related to adsorption capacity; 1/n is the Freundlich exponent indicating that whether the adsorption process is favorable or not. Generally, when the value of n is larger than 2, the adsorption system could be considered as a favorable process. The slope 1/n also represents the adsorption intensity of surface heterogeneity. As the value of 1/n gets closer to 0, the adsorption system is becoming more heterogeneous [44]. As represented in Fig. 5, the adsorption capacity of pristine PAC and PCB-PAC increased with the increasing initial concentrations. This phenomenon was caused by the increasing driving force generated by higher MB concentrations which could make the adsorbate overcome the mass transfer resistance from aqueous phase to adsorbent phase [45]. After achieving saturation, no more MB could be adsorbed on the adsorbents. Different regression methods of nonlinear and linear processes had been used to simulate the experimental data that would be more rational and reliable to elucidate the adsorption process [46]. The corresponding parameters were calculated and summarized in Table 3. According to these results, the adsorption data of MB on pristine PAC fitted Freundlich isotherm model better than Langmuir isotherm model, indicating the adsorption process mainly occurred on heterogeneous surface. Here, the value of 1/n by pristine PAC was much closer to 0 than PCB-PAC validating that the adsorption process of PAC was carried out in a heterogeneous system. The results were consistent with the phenomenon that the PAC tended to aggregate especially in viscous and complex solutions like blood or plasma. For PCB-PAC, the Langmuir model was a better fitting isotherm for the experimental data. The results indicated that the adsorption process took place on a homogeneous surface and PAC particles were homogenously distributed in the PCB hydrogel matrix. For all used initial concentrations of MB, the values of RL for PCB-PAC were raging between 0.033 and 0.1021, indicating that the adsorption process was favorable and PCB-PAC was a preferred adsorbent. It could be obtained from the experimental data that the qm of MB by pristine PAC was about 400 mg/g. The qm of PCB-PAC was about 330 mg/g, calculated by Langmuir model
and was not significantly reduced compared with pristine PAC.
3.5. Hemolysis analysis Blood-contact material with poor hemocompatibility can cause the disruption of the erythrocyte membrane and consequently the release of hemoglobin into the plasma, known as hemolysis. For medical applications, the HR value of the blood-contacting materials must be below 5% [47]. The results of hemolysis assay are shown in Fig. 6. The supernatant of the negative control (0.9% NaCl solution) showed no visible hemolysis effect. In comparison, the pristine PAC sample showed a significant hemolysis effect and the HR was 14.82%. All the hydrogel coated PAC samples demonstrated a much lower degree of hemolysis (<1%); while PCB-PAC performed best with the lowest hemolysis value (0%).
3.6. Adsorption in biological fluids BSA solution and 100% FBS were used to evaluate the adsorption performance of PCB-PAC in the clinical mimetic situations. As shown in Fig. 7a-c, photo images showed the color changes in different solutions before and after incubated with pristine PAC and PCB-PAC. It appeared that pristine PAC could efficiently remove MB in PBS solution but significantly lost its adsorption ability at the present of BSA or serum proteins. This was consistent with literature reports and could be expected because pristine PAC could be easily coated by proteins and their active sites got blocked [3, 48]. As shown in Fig. 7d, pristine PAC quickly reached equilibrium (1 h) and more than 99% of MB was removed in PBS; however, it took more than 7 h to reach equilibrium in BSA solution or in 100% FBS. 86% of MB was removed in BSA solution, and only less than 60% adsorption was achieved in 100% FBS. In comparison, protected by the antifouling PCB hydrogel coating, PCB-PAC could efficiently resist protein adsorption even in 100% serum. As result, its adsorption performance was not compromised as shown in Fig. 7e. It took similar amount of time to reach equilibrium and more than 95% of MB was removed. It could be advantageous for the patients in hemoperfusion to avoid the hemoperfusion-related complications. These results suggested that the PCB hydrogel could be suitable as the coating material for the improvement of toxin clearance from blood.
3.7. Adsorption of bilirubin from albumin A major advantage of hemoperfusion over other blood purification techniques is the ability to isolate also molecules bound to plasma proteins. Therefore, we also tested BR, a very commonly studied pathogenic toxin, as another model molecule. BR is a lipophilic molecule that can strongly bind to albumin (Ka= 9.5×107 M-1) via one high affinity site and one low affinity binding site [38, 49]. The amount of unbound BR fraction in the equilibrium is about 0.1% [5]. As shown in Fig. 8, PCB-PAC reached equilibrium after 2.5 h and the adsorption capability was 8 mg/g, while pristine PAC completely lost its adsorption ability. This result indicated that BR could be detached from the BR-BSA complexes and adsorbed by PCB-PAC during the process.
Therefore, we proved that PCB-PAC also possessed the ability to isolate molecules that can strongly bind to plasma proteins.
4. Conclusions In this study, we developed PCB hydrogel coated PAC to improve its hemocompatibility in hemoperfusion applications. The large mesh size of the PCB hydrogels allowed efficient mass transfer of the adsorbates, and was superior to PEGMA or PHEMA hydrogels which were commonly used. The results of adsorption isotherms indicated that the PCB hydrogel coating had not significantly decreased the equilibrium adsorption capacity of PAC. Most importantly, the antifouling PCB hydrogel coating significantly improved the hemocompatibility of PAC: on one hand, PCB-PAC showed negligible hemolysis; on the other hand, PCB coating could efficiently resist the nonspecific protein adsorption even in 100% serum solution and maintain the adsorption performance of the encapsulated PAC. In addition, PCB-PAC also had the ability to isolate and adsorb protein-binding toxin. Findings of this work pave the way of high efficient hemoperfusion adsorbents and may significantly improve many different clinical hemoperfusion therapies.
Acknowledgements The authors acknowledge the financial support from the National Natural Science Funds for Excellent Young Scholars 21422605, the National Natural Science Funds for Innovation Research Groups 21621004, the Tianjin Natural Science Foundation, 14JCYB-JC41600, the Research Fund for the Doctoral Program of Higher Education of China 20130032120089, and the Program for New Century Excellent Talents in University NCET-13-0417.
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Scheme. 1. Chemical structures of PEGMA, PHEMA and PCB.
Fig. 1. Encapsulation stability measured as optical density (600 nm) of the solution in contact with hydrogel coated PAC after 3 subsequent mechanical shaking incubations with 0.9% NaCl solution containing heparin (300 units/mL).
Fig. 2. The removal efficiency of MB by PAC and hydrogel coated PAC (adsorption performed in PBS, initial concentration of MB 50 mg/L).
Fig. 3. Pseudo-1st-order (a) and Pseudo-2nd-order (b) models for the adsorption of MB by PAC and PCB-PAC.
Fig. 4. Intraparticle diffusion model for the adsorption of MB by PAC and PCB-PAC.
Fig. 5. Adsorption isotherms of MB by PAC and PCB-PAC fitted by different regression methods including the Langmuir model (a, b) and Freundlich model (c, d).
Fig. 6. Hemolysis assay for PAC and hydrogel coated PAC.
Fig. 7. Adsorption performance of MB by PAC and PCB-PAC in biological fluids (initial concentration of MB 50 mg/L). (a) is the adsorption of MB by PAC and PCB-PAC in PBS. (b) is the adsorption of MB by PAC and PCB-PAC in BSA solution. (c) is the adsorption of MB by PAC and PCB-PAC in 100% FBS. (d) is the adsorption of MB by PAC in PBS, BSA solution and 100% FBS. (e) is the adsorption of MB by PCB-PAC in PBS, BSA solution and 100% FBS.
Fig. 8. The adsorption capacity of BR by PAC and PCB-PAC (adsorption performed in 40 mg/mL BSA solution, initial concentration of BR 150 mg/L).
Table 1 Physical properties of adsorbents. Samples
Swelling Degree
Compressive modulus (MPa)
Break Strain (%)
Mesh Size (nm)
PCB
1.688±0.013
0.067±0.005
61.173±0.008
5.598
PCB-PAC
1.717±0.012
0.074±0.004
71.318±0.001
——
PEGMA
1.121±0.041
0.445±0.012
28.898±0.006
2.264
PEGMA-PAC
1.174±0.052
0.526±0.017
31.280±0.005
——
PHEMA
0.789±0.032
0.264±0.011
——
1.943
PHEMA-PAC
0.831±0.044
0.358±0.015
——
——
Table 2 Kinetic constants for pseudo-first model (K1 min-1), pseudo-second order model (K2 g mg-1 min-1; qe mg/g) and intraparticle diffusion model (K3 mg g-1 min1/2) for adsorption of MB by PAC and PCB-PAC. Pseudo-1st-order Adsorbents
K1
R12
PAC
0.1811
0.2951
PCB-PAC
0.2354
0.6569
Pseudo-2nd-order
Intraparticle diffusion
qe.exp
qe.cal
R22
3.6101
99.49
100.00
0.9997
0.2963
95.202
0.9992
0.3537
99.19
103.09
0.9984
3.4322
39.643
0.9923
K2×10
3
K3
C
Table 3 Langmuir isotherm model (q m mg/g; KL L/mg) and Freundlich isotherm model (KF (mg/g)(L/mg) 1/n) constants and correlation coefficients for the adsorption of MB by PAC and PCB-PAC. Adsorbents PAC PCB-PAC
Fitting method
Langmuir
Freundlich 2
qm
KL
R
KF
1/n
R2
Linear Non-linear
366.3004
0.8273
0.7463
213.0591
0.1320
0.9715
380.5284
0.6080
0.6686
213.4364
0.1392
0.9569
Linear Non-linear
327.8689
0.07325
0.9771
119.2889
0.1863
0.9175
324.5723
0.08092
0.9722
124.8735
0.1810
0.9068
R32
S0021-9797(17)30417-4 http://dx.doi.org/10.1016/j.jcis.2017.04.024 YJCIS 22236
To appear in:
Journal of Colloid and Interface Science
Received Date: Revised Date: Accepted Date:
24 February 2017 6 April 2017 7 April 2017
Please cite this article as: N. Cai, Q. Li, J. Zhang, T. Xu, W. Zhao, J. Yang, L. Zhang, Antifouling zwitterionic hydrogel coating improves hemocompatibility of activated carbon hemoadsorbent, Journal of Colloid and Interface Science (2017), doi: http://dx.doi.org/10.1016/j.jcis.2017.04.024
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Graphical abstract
Antifouling zwitterionic hydrogel coating improves hemocompatibility of activated carbon hemoadsorbent Nana Cai,a,b,c Qingsi Li,a,b,c Jiamin Zhang,a,b,c Tong Xu,a,b,c Weiqiang Zhao,a,b,c Jing Yanga,b,c and Lei Zhang* a,b,c a Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China b Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin, 300072, PR China c Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin, 300072, PR China Corresponding author at: Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China. E-mail address: [email protected] Cell: 86-13502038015
Abstract Activated carbon has been widely used in hemoperfusion treatments. However, its performance has been significantly compromised by their poor hemocompatibility. In this work, we developed a novel antifouling adsorbent based on zwitterionic poly-carboxybetaine (PCB) hydrogel and powdered activated carbon (PAC) to improve hemocompatibility. We found this new adsorbent (PCB-PAC) was highly stable with negligible leakage of activated carbon debris. It could efficiently resist protein adsorption and avoid any hemolysis effect. The adsorption performance of PCB-PAC for methylene blue was not influenced in a single protein solution or even in 100%
fetal bovine serum (FBS), in which pristine PAC lost 50% of its adsorption ability. The isotherms results showed that the adsorption process of PCB-PAC fitted the Langmuir isotherm well, indicating that the PAC particles were homogenously distributed in the PCB hydrogel matrix. Moreover, PCB-PAC could also adsorb bilirubin molecules bound to albumin in solution, while pristine PAC showed no discernible adsorption effect. Findings in this work hold great potential to significantly improve the performance and efficiency of current extracorporeal devices for removing toxins from blood directly.
Keywords: zwitterionic hydrogel anti-biofouling activated carbon adsorption hemocompatibility
Introduction Basing on adsorption, hemoperfusion (HP) technology can be used to remove toxic substances from the blood of patients, thus it has been widely used clinically in the treatment of severe intoxications, nephritic, hepatic/multiorgan insufficiency, autoimmune diseases, sepsis, etc [1-3]. Unlike hemodialysis (HD) and hemofiltration (HF), which mainly remove small and hydrophilic molecules from blood, a unique ability of hemoperfusion is to efficiently adsorb molecules with larger molecular weight and/or high protein-binding affinity [4-6]. The curative efficiency of hemoperfusion critically depends on the performance and hemocompatibility of the adsorbents [3, 7]. Therefore, the development of high efficient and hemocompatible adsorbents is the main focus in hemoperfusion research and development [8]. Activated carbon is the most commonly used material as a nonspecific adsorbent in a clinical setting due to its strong adsorption ability and low cost [9-11]. However, pristine activated carbon particles present poor blood compatibility which hinders their successful practical applications: firstly, upon contacting with blood, they suffer from non-specific blood protein adsorption which would cover the adsorbents’ surface and significantly reduce their adsorption ability; secondly, activated carbon materials would result in serious clinical side effects such as particulate microemboli formation, hemolysis and blood coagulation [12-14]. To solve this problem, Chang et al. introduced a microencapsulation process that could cover the activated carbon particles with a polymeric coating [15]. In this way, the coating could directly contact with blood instead of bare activated carbon, thus the hemocompatibility and the hemoperfusion performance could be improved [16]. In the pioneering studies of Chang, a number of coating materials such as cellulose nitrate, albumin-collodion, cellulose acetate, and polyamide have been evaluated [17-19]. Later, Andrade et al. investigated the use of hydrogel as coating material for activated carbon particles [20]. Hydrogel is a crosslinked hydrophilic polymeric network with high water content and good biocompatibility [21]. It could ensure better diffusive permeability and provide biomimetic mechanical strengths [22-24]. These work lead to the commercialization of polymer coatings (cellulose acetate, polyhydroxyethylmethacrylate, polyacrylic, etc) based activated carbon adsorbents [13]. However, current coating materials still could not fully
eliminate the negative influence in hemoperfusion applications and therefore the development of novel polymer coatings with better performance is still highly desired [14, 16].
Ideal coating materials for activated carbon must possess excellent blood compatibility without hemolysis, protein adsorption or coagulation, while should also be highly permeable to target toxins [16, 25]. Recently, zwitterionic materials have received increasingly attention in many biomedical applications, attributed to their excellent “antifouling” properties to resist non-specific protein adsorption as well as cell or bacteria adhesion. Owing to the opposing but balanced charges in each moiety, zwitterionic-based materials can strongly bind water molecules via electrostatically induced hydration, which poses an obstacle for protein settlement. Moreover, zwitterionic hydrogels exhibit high water content and large mesh size which could benefit the diffusion of molecules through the hydrogel and provide biomimetic mechanical strengths [23, 26]. Among zwitterionic materials, carboxybetaine (CB) is a very interesting and unique one. The structure of CB monomer is similar to glycine betaine, a natural compound in the human body for osmotic regulation. PCB coated surface could reduce protein adsorption to an undetectable level even in 100% human blood serum [26]. Moreover, PCB possess many unique properties: Zhang et al. in 2008 reported that polyCBMA exhibited unique anticoagulant activity which made it a promising polymer for blood-contacting applications [27]. In 2012, Hsiu-Wen Chien et al. proved that PCB hydrogel was highly compatible with living cells and could maintain the viability of the encapsulated cells. This also proved the excellent permeability of PCB hydrogel to nutrients and cell wastes [28]. In more recent studies, PCB hydrogel was shown to resist the foreign body reaction when implanted in mice and effectively enhance protein stability [29-31]. These findings motivated us to explore the application of PCB hydrogel as the coating material for activated carbon particles. In the present study, we developed a novel adsorbent for hemoperfusion based on PCB hydrogel and PAC particles. We demonstrated that the new adsorbent was highly hemocompatible with fresh blood and its adsorbing ability was not compromised even in 100% blood serum. Furthermore, the adsorption of bilirubin was investigated to show the versatile adsorption abilities of the PCB-PAC. The results showed that PCB-PAC would be a potential biomedical material for detoxification of biological fluids, in particular blood or blood plasma of patients.
2. Materials and methods 2.1. Materials Methylene blue (MB) was purchased from J&K Scientific Ltd. (Beijing, China). Bovine serum albumin (BSA) and bilirubin (BR) were purchased from Solarbio Life Science. Fetal bovine serum (FBS) was obtained from Gibco. Ammonium persulfate (APS) and N,N,N’,N’-Tetramethylethylenediamine (TEMED) were purchased from Across Organics. N,N’-Methylenebis (acrylamide) (MBAA) was purchased from Alfa Aesar. Tetraethylene glycol dimethacrylate (TEGDMA) and Sodium chloride were p-
urchased from Sinopharm Chemical Reagent Bejing Co., Ltd. (Beijing, China). Phosphate buffer saline (PBS) solution (0.01 M, pH 7.4) was made from PBS powder purchased from Solarbio Life Science. Ethanol, ethylene glycol and sodium hydroxide were purchased from Yuanli, Tianjin. Milli-Q water (18.2 MΩ cm-1) was used in all experiments. 2-Hydroxyethyl methacrylate (HEMA) was purchased from Acros Organics. Poly(ethylene glycol) methacrylate (PEGMA Mn 500) was obtained from Sigma-Aldrich. Carboxybetaine methacrylate (CBMA) was synthesized as previously reported [23, 32].
2.2. Adsorbents preparation The granular activated carbon was grinded to microparticles and then sieved to remove particles greater than 50 microns. To prepare PCB-PAC, CBMA monomer and MBAA crosslinker were dissolved in 1 M NaCl at a concentration of 40% (wt/vol) and a crosslinking ratio of 1% (molar percent of monomer) [33]. The solution was homogenized by sonication in an ice bath for 30 min. Then, PAC particles were added at 160 mg per 1 mL solution. After PAC addition, the mixture was stirred overnight and then sonicated for 10 min to obtain a homogenous slurry. After this, 1 mg of the initiators (APS) and 1 μL the promoters (TEMED) were added to the solution to initiate polymerization between glass microscope slides separated by 0.5 mm thick polytetrafluoroethylene (PTFE) spacers at 37 ℃ for 30 min. The sample was then removed from the slides and immersed in PBS for equilibration. The buffer was changed every 12 hours for 5 days. [23, 32, 34] Similarly, to prepare PHEMA-PAC, HEMA monomer and the crosslinker (TEGDMA) were added to a mixed solvent (ethanol/ethylene glycol/H2O =1:1.5:1.5, volume ratio) at a concentration of 40% and a crosslinking ratio of 1% [35]. Then, PAC particles (170 mg/mL) were added and the solution was stirred overnight and sonicated well. Finally, 2 mg of the initiators and 1.5 μL the promoters were added to initiate the polymerization as described above. PEGMA-PAC was prepared by mixing PEGMA monomer and the crosslinker (TEGDMA) to ethylene glycol at a concentration of 40% and a crosslinking density of 1% [36]. Then, PAC particles (167 mg) were added to the solution (1 mL) and the mixture was stirred overnight and sonicated well. Finally, 3 mg of the initiators and 2 μL the promoters were added to the mixed solution to initiate the polymerization as described above. Pure PCB, PHEMA and PEGMA hydrogels without PAC were prepared as control samples. All samples were cut into 1 mm-sized pellets and washed with PBS before use.
2.3. Characterizations 5 mm diameter disks of all hydrogel samples with or without PAC were tested using a WDW-5 electromechanical tester (Beijing Time Technologies CO., LTD, China) with a 200 N load cell at a rate of 0.5 mm/min. The Young’s modulus was calculated from 10% to 20% strain. The equilibrium swelling degree of the samples was performed by calculating the
volume ratio of samples after and before swelling. The diameter and thickness of the fully swollen disks were measured using the vernier caliper. The equilibrium swelling degree (SD) was determined using the following equation:
(2-1) Where Vs and Vr are the volumes of the fully swollen and the unswollen disks; DS and DR are the diameters of the fully swollen and the unswollen disks; Ls and Lr are the thicknesses of fully swollen and unswollen disks, respectively. According to the swelling degree and mechanical properties, mesh size of the hydrogel coatings (distance between crosslinks) can be determined by the following two equations:
(2-2) Where is the stress at a particular strain, in units of Pa, α is the deformation ratio related to the stress determined as the ratio of elastically deformed length L to initial length L0 of a crosslinked hydrogel under compression. R is the universal gas constant, T is absolute temperature, 2 is the volume fraction of polymer at equilibrium (in the fully swollen hydrogel), and o is the volume fraction of polymer in the relaxed state (non-swollen, but not dehydrated hydrogel).
(2-3) where NA is Avagadro’s number. The value of hydrogel coatings. [23]
is definitely mesh size of the
2.4. Stability tests The encapsulation stability of the PAC microparticles in different hydrogels was evaluated by incubating the samples in 1 mL 0.9% NaCl solution containing 300 units of injectable heparin sodium salt according to the previous report [14]. The mixtures were incubated in a shaking water bath at 175 rpm, 25 ℃ for 60 min and then the optical density of the supernatant was measured at 600 nm for the leakage of fine microparticles. This experiment was repeated three times by adding fresh solution each time.
2.5. Adsorption tests To study the adsorption behavior of different hydrogel coated absorbents, PCB-PAC, PEGMA-PAC and PHEMA-PAC samples (all containing 10 mg PAC) were placed into flasks containing 20 mL of 50 mg/L MB in PBS solution. 10 mg pristine PAC was studied as the control sample. The flasks were placed in a shaking water bath of 175 rpm at 25 ℃. The residual concentrations were measured at different time points using ultraviolet-visible (UV-Vis) absorption spectroscopy at a wavelength of 663 nm.
The removal efficiency (%) was determined using the following equation:
(2-4) where Re, C0 and Ct are the removal efficiency of MB by the adsorbents at time t (%), the initial MB concentration (mg/L) and the residual MB concentration at time t (mg/L), respectively.
2.6. Adsorption isotherms The MB adsorption capacity of the pristine PAC and the PCB-PAC was investigated by batch adsorption experiments with MB in PBS solution. 10 mg pristine PAC and PCB-PAC (containing 10 mg PAC) were equilibrated in 20 mL solutions with different MB concentrations in a shaking water bath of 175 rpm at 25 ℃ for 24 h. The amount of adsorbed MB was calculated from the residual amount of MB in the solution via the mass balance. The amount of MB adsorbed at equilibrium qe (mg/g) was calculated according to the following equation:
(2-5) In this equation, qe is the amount of solute adsorbed per unit weight of the PAC particles at equilibrium (mg/g) and Ce is equilibrium concentration of MB in solution after adsorption (mg/L).
2.7. Hemolysis evaluation In this work, the hemolysis effect was evaluated by incubation of the hydrogel coated PAC and pristine PAC in diluted blood containing 20% fresh blood and 80% normal saline at 37 ℃ for 30 min. Blood samples diluted with 80% normal saline and deionized water in the absence of adsorbents were used as negative control and positive control, respectively. Then, the blood samples were centrifuged at 1000 rpm for 10 min and the colors of the supernatants were evaluated. The hemolysis ratio (HR) was determined by measuring the absorbance of supernatants at 541 nm by UV-Vis spectroscopy and calculated with the following equation: (2-6) Where AS, AN and AP are the absorbance of the samples, the negative control and the positive control, respectively. [37]
2.8. Performance in biological fluids To test the adsorption performance of pristine PAC and PCB-PAC in biological fluids, MB was dissolved in PBS solution, PBS solution containing 40 mg/mL BSA and 100% FBS. 10 mg pristine PAC and PCB-PAC (containing 10 mg PAC) were incubated with 20 mL of MB in different solutions with a same initial concentration of 50 mg/L. The samples (0.1 mL) were taken at different time points and were analyzed the
residual concentrations. The removal efficiency at different time points was measured using the equation described above.
2.8. Adsorption of bilirubin from albumin To evaluate the ability of PCB-PAC to isolate molecules bound to plasma proteins, bilirubin-albumin (BR-BSA) solution was prepared by dissolving solid bilirubin in 0.1 M NaOH, then adding the resulting solution into BSA solution, and the final pH was adjusted to 7.4 [38]. PCB-PAC particles (containing 30 mg PAC for each test) were placed into flasks containing 5 mL BR-BSA solution (150 mg/L BR, 40 mg/mL BSA). 30 mg pristine PAC was used as the control sample. The flasks were placed in a shaking water bath of 175 rpm at 25 ℃. The whole procedure was conducted in the dark to avoid photodegradation of BR. The residual concentrations were measured at different time points using ultraviolet-visible (UV-Vis) absorption spectroscopy at a wavelength of 460 nm. The equilibrium adsorption amount of BR qe (mg/g) was measured using the equation described above.
3. Results and Discussion 3.1. Physical properties of adsorbents The physical properties of the samples are summarized in Table 1. The compressive modulus and the break strain of the hydrogel coated PAC samples were slightly higher than those of the corresponding pure hydrogels. The PCB-PAC possessed larger break strain (71%) than PEGMA-PAC (31%), indicating PCB-PAC could better maintain their integrity during the adsorption process. In contrast, PEGMA-PAC displayed a higher compressive modulus but a lower break strain. The hydrogel coated PAC samples exhibited the similar swelling degree as that of the pure hydrogels. Due to the strong hydration properties, PCB hydrogel coating possessed the largest swelling degree (1.69), while that of PEGMA hydrogel coating was 1.12. In comparison, PHEMA hydrogel coating showed much lower swelling degree (0.79), which meant it slightly shrank in aqueous solutions. Larger mesh size allowed better mass transfer for molecules through the hydrogel coating which was crucial in hemoperfusion applications. For PEGMA and PHEMA hydrogel coatings, the mesh size distributions were narrow and the diameters were 2.26 nm and 1.94 nm, respectively. Attributed to the largest swelling degree, the mesh size of PCB hydrogel coating (5.59 nm) was twice more than those of PEGMA and PHEMA hydrogel coatings which ensures higher diffusive permeability.
3.2. Adsorbents stability One important advantage of the hydrogel encapsulation method is to avoid the leakage of PAC debris, which may cause severe side effects clinically. The stability tests (Fig. 1) showed that PCB-PAC was highly stable with negligible release of fine
PAC microparticles thanks to its high break strain and good stability. In comparison, PEGMA-PAC and PHEMA-PAC were much less stable and showed obvious PAC microparticles leakage. Due to the poor mechanical strength, PEGMA-PAC tended to collapse under shaking, while although PHEMA hydrogel was much stronger, the encapsulation appeared unstable possibly due to the poor compatibility between PHEMA hydrogel and the PAC particles.
3.3. Adsorption properties MB (molecular weight 319.86 Da) was chosen as a model molecule. It has been commonly used to evaluate the adsorption performance of activated carbon in aqueous solutions and study the effects of coating materials on adsorption behaviors [2, 39, 40]. All three types of hydrogel coated PAC samples were prepared as 1 mm-sized particles. The surface area of hydrogel particles (containing 10 mg PAC in each test) was about 10-4 m2, while the surface area of 10 mg PAC was about 12 m2, which was much larger and dominated the adsorption capacity. The different adsorption behaviors of the samples are shown in Fig. 2. It could be seen that all three types of hydrogel coated PAC samples showed a decreased adsorption profile compared with pristine PAC. This was because the hydrogel coatings performed as mass transfer barriers for MB. However, thanks to the large mesh size of PCB hydrogel coating, the adsorption performance of PCB-PAC was not significantly influenced. In comparison, mesh sizes of PHEMA and PEGMA hydrogels were much smaller, thus their adsorption rates were delayed and their adsorption capacities were significantly decreased. The PCB-PAC removed almost 100% of MB after 7.5 h, similar to pristine PAC, while the removal ratios of PEGMA-PAC and PHEMA-PAC were only 52% and 31%, respectively. This was possibly caused by a large ratio of adsorption sites in PAC were made inaccessible by the compact structure of PEGMA and PHEMA hydrogels. Therefore, PCB-PAC was found to be the most suitable adsorbent among the three samples tested. To further understand the adsorption process of PCB-PAC, we investigated the adsorption kinetics and isotherms in PBS solution. Two widely used kinetic models, pseudo-first-order model (Eq. 3-1) and pseudo-second-order model (Eq. 3-2), were used to study the adsorption kinetic behaviors of MB on pristine PAC and PCB-PAC [41, 42]. (3-1)
(3-2) In Eq. (3-1), qt is the amount of MB adsorbed per unit weight of PAC particles at time t (mg/g); k1 is the rate constant of pseudo-first-order model (min-1). In Eq. (3-2), k2 is the rate constant of pseudo-second-order model (g mg-1 min-1); h=k2qe2 is the initial sorption rate (mg g-1 min-1). The fitting results using pseudo-first-order model and pseudo-second-order model are shown in Fig. 3. The rate constants and the correlation coefficients related to each
model were calculated and listed in Table 2. All the experimental data showed better compliance with pseudo-second-order model in terms of higher correlation coefficient values (R2>0.998). The experimental qe values (qe.exp) showed a very good agreement with the theoretical ones (qe.cal), calculated by pseudo-second-order model. The initial adsorption rate (h= k2qe2) obtained from this model validated the experimental removal results that the pristine PAC had a faster initial adsorption rate than the PCB-PAC under similar conditions. This model was valid for pristine PAC and PCB-PAC which could be utilized to describe the adsorption kinetics and to forecast the adsorption capacity at t (time). The intraparticle diffusion model (Eq. 3-3) was used to elucidate the diffusion mechanism during the adsorption process [43]. (3-3) In Eq. (3-3), K3 and C are the rate constant of the intraparticle diffusion model (mg g-1 min1/2) and the intercept, respectively. The value of C gives an idea about the thickness of the boundary layer. A larger value of C represents a greater boundary layer diffusion effect. If the value of C is 0, the intraparticle diffusion is the sole rate controlling step. If the value of C is not 0, the intraparticle diffusion is not the only rate controlling step and other factors may also control the process of adsorption. As shown in Fig. 4, the removal of MB was found to be rapid at the initial period which was controlled by the external surface diffusion of adsorbate or the boundary layer diffusion. The adsorption behaviors were followed by a gradual adsorption stage where intraparticle diffusion was rate limiting. The final stage was achieved equilibrium of which the intraparticle diffusion started to slow down due to the extremely low MB concentration left in the solution. [43] The corresponding parameters are calculated and summarized in Table 2. It was found that the intraparticle diffusion was not the only rate controlling step in the adsorption process of pristine PAC and PCB-PAC (C≠0). The pristine PAC showed an initial fast adsorption stage and then reached equilibrium. These results indicated that a significant boundary layer diffusion effect occurred in the adsorption process of pristine PAC. It was consistent with the results in literatures which had shown that the boundary layer diffusion was primary rate controlling step in small size adsorbents [43]. Whereas in the adsorption process of PCB-PAC, the diffusion of molecules through the polymeric matrix into the PAC particles was the primary rate controlling step. This adsorption behavior was well correlated with the structure of the PCB-PAC.
3.4. Adsorption Isotherms The correlation of the equilibrium adsorption capacity and equilibrium concentration can be represented by the adsorption isotherms. Two models are commonly used for studies of adsorption, the Langmuir isotherm (Eq. 3-4) and the Freundlich isotherm (Eq. 3-6). This experiment through the modeling analysis provided an insight into the adsorption mechanism and surface properties of adsorbents. The Langmuir isotherm is suitable in homogeneous systems in which all adsorbing sites possess equal affinity for the adsorbate, while the Freundlich model refers to multilayer adsorption indicati-
ng non-uniform distribution of binding affinities over the heterogeneous systems. The essential characteristics of the Langmuir isotherm can be described by a separation factor RL (Eq. 3-5). Non-Linear form:
Non-Linear form:
Linear form:
(3-4)
（
(3-5)
Linear form:
(3-6)
In Eq. (3-4), qm is the maximum adsorption capacity of adsorbent (mg/g); KL is the Langmuir constant associated to the affinity of binding sites. In Eq. (3-5), RL is the separation factor indicating that the adsorption process is favorable or not. According to this model, when RL is between 0 and 1, the adsorption system is favorable. In Eq. (3-6), KF (mg/g)(L/mg)1/n is the Freundlich constant related to adsorption capacity; 1/n is the Freundlich exponent indicating that whether the adsorption process is favorable or not. Generally, when the value of n is larger than 2, the adsorption system could be considered as a favorable process. The slope 1/n also represents the adsorption intensity of surface heterogeneity. As the value of 1/n gets closer to 0, the adsorption system is becoming more heterogeneous [44]. As represented in Fig. 5, the adsorption capacity of pristine PAC and PCB-PAC increased with the increasing initial concentrations. This phenomenon was caused by the increasing driving force generated by higher MB concentrations which could make the adsorbate overcome the mass transfer resistance from aqueous phase to adsorbent phase [45]. After achieving saturation, no more MB could be adsorbed on the adsorbents. Different regression methods of nonlinear and linear processes had been used to simulate the experimental data that would be more rational and reliable to elucidate the adsorption process [46]. The corresponding parameters were calculated and summarized in Table 3. According to these results, the adsorption data of MB on pristine PAC fitted Freundlich isotherm model better than Langmuir isotherm model, indicating the adsorption process mainly occurred on heterogeneous surface. Here, the value of 1/n by pristine PAC was much closer to 0 than PCB-PAC validating that the adsorption process of PAC was carried out in a heterogeneous system. The results were consistent with the phenomenon that the PAC tended to aggregate especially in viscous and complex solutions like blood or plasma. For PCB-PAC, the Langmuir model was a better fitting isotherm for the experimental data. The results indicated that the adsorption process took place on a homogeneous surface and PAC particles were homogenously distributed in the PCB hydrogel matrix. For all used initial concentrations of MB, the values of RL for PCB-PAC were raging between 0.033 and 0.1021, indicating that the adsorption process was favorable and PCB-PAC was a preferred adsorbent. It could be obtained from the experimental data that the qm of MB by pristine PAC was about 400 mg/g. The qm of PCB-PAC was about 330 mg/g, calculated by Langmuir model
and was not significantly reduced compared with pristine PAC.
3.5. Hemolysis analysis Blood-contact material with poor hemocompatibility can cause the disruption of the erythrocyte membrane and consequently the release of hemoglobin into the plasma, known as hemolysis. For medical applications, the HR value of the blood-contacting materials must be below 5% [47]. The results of hemolysis assay are shown in Fig. 6. The supernatant of the negative control (0.9% NaCl solution) showed no visible hemolysis effect. In comparison, the pristine PAC sample showed a significant hemolysis effect and the HR was 14.82%. All the hydrogel coated PAC samples demonstrated a much lower degree of hemolysis (<1%); while PCB-PAC performed best with the lowest hemolysis value (0%).
3.6. Adsorption in biological fluids BSA solution and 100% FBS were used to evaluate the adsorption performance of PCB-PAC in the clinical mimetic situations. As shown in Fig. 7a-c, photo images showed the color changes in different solutions before and after incubated with pristine PAC and PCB-PAC. It appeared that pristine PAC could efficiently remove MB in PBS solution but significantly lost its adsorption ability at the present of BSA or serum proteins. This was consistent with literature reports and could be expected because pristine PAC could be easily coated by proteins and their active sites got blocked [3, 48]. As shown in Fig. 7d, pristine PAC quickly reached equilibrium (1 h) and more than 99% of MB was removed in PBS; however, it took more than 7 h to reach equilibrium in BSA solution or in 100% FBS. 86% of MB was removed in BSA solution, and only less than 60% adsorption was achieved in 100% FBS. In comparison, protected by the antifouling PCB hydrogel coating, PCB-PAC could efficiently resist protein adsorption even in 100% serum. As result, its adsorption performance was not compromised as shown in Fig. 7e. It took similar amount of time to reach equilibrium and more than 95% of MB was removed. It could be advantageous for the patients in hemoperfusion to avoid the hemoperfusion-related complications. These results suggested that the PCB hydrogel could be suitable as the coating material for the improvement of toxin clearance from blood.
3.7. Adsorption of bilirubin from albumin A major advantage of hemoperfusion over other blood purification techniques is the ability to isolate also molecules bound to plasma proteins. Therefore, we also tested BR, a very commonly studied pathogenic toxin, as another model molecule. BR is a lipophilic molecule that can strongly bind to albumin (Ka= 9.5×107 M-1) via one high affinity site and one low affinity binding site [38, 49]. The amount of unbound BR fraction in the equilibrium is about 0.1% [5]. As shown in Fig. 8, PCB-PAC reached equilibrium after 2.5 h and the adsorption capability was 8 mg/g, while pristine PAC completely lost its adsorption ability. This result indicated that BR could be detached from the BR-BSA complexes and adsorbed by PCB-PAC during the process.
Therefore, we proved that PCB-PAC also possessed the ability to isolate molecules that can strongly bind to plasma proteins.
4. Conclusions In this study, we developed PCB hydrogel coated PAC to improve its hemocompatibility in hemoperfusion applications. The large mesh size of the PCB hydrogels allowed efficient mass transfer of the adsorbates, and was superior to PEGMA or PHEMA hydrogels which were commonly used. The results of adsorption isotherms indicated that the PCB hydrogel coating had not significantly decreased the equilibrium adsorption capacity of PAC. Most importantly, the antifouling PCB hydrogel coating significantly improved the hemocompatibility of PAC: on one hand, PCB-PAC showed negligible hemolysis; on the other hand, PCB coating could efficiently resist the nonspecific protein adsorption even in 100% serum solution and maintain the adsorption performance of the encapsulated PAC. In addition, PCB-PAC also had the ability to isolate and adsorb protein-binding toxin. Findings of this work pave the way of high efficient hemoperfusion adsorbents and may significantly improve many different clinical hemoperfusion therapies.
Acknowledgements The authors acknowledge the financial support from the National Natural Science Funds for Excellent Young Scholars 21422605, the National Natural Science Funds for Innovation Research Groups 21621004, the Tianjin Natural Science Foundation, 14JCYB-JC41600, the Research Fund for the Doctoral Program of Higher Education of China 20130032120089, and the Program for New Century Excellent Talents in University NCET-13-0417.
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Scheme. 1. Chemical structures of PEGMA, PHEMA and PCB.
Fig. 1. Encapsulation stability measured as optical density (600 nm) of the solution in contact with hydrogel coated PAC after 3 subsequent mechanical shaking incubations with 0.9% NaCl solution containing heparin (300 units/mL).
Fig. 2. The removal efficiency of MB by PAC and hydrogel coated PAC (adsorption performed in PBS, initial concentration of MB 50 mg/L).
Fig. 3. Pseudo-1st-order (a) and Pseudo-2nd-order (b) models for the adsorption of MB by PAC and PCB-PAC.
Fig. 4. Intraparticle diffusion model for the adsorption of MB by PAC and PCB-PAC.
Fig. 5. Adsorption isotherms of MB by PAC and PCB-PAC fitted by different regression methods including the Langmuir model (a, b) and Freundlich model (c, d).
Fig. 6. Hemolysis assay for PAC and hydrogel coated PAC.
Fig. 7. Adsorption performance of MB by PAC and PCB-PAC in biological fluids (initial concentration of MB 50 mg/L). (a) is the adsorption of MB by PAC and PCB-PAC in PBS. (b) is the adsorption of MB by PAC and PCB-PAC in BSA solution. (c) is the adsorption of MB by PAC and PCB-PAC in 100% FBS. (d) is the adsorption of MB by PAC in PBS, BSA solution and 100% FBS. (e) is the adsorption of MB by PCB-PAC in PBS, BSA solution and 100% FBS.
Fig. 8. The adsorption capacity of BR by PAC and PCB-PAC (adsorption performed in 40 mg/mL BSA solution, initial concentration of BR 150 mg/L).
Table 1 Physical properties of adsorbents. Samples
Swelling Degree
Compressive modulus (MPa)
Break Strain (%)
Mesh Size (nm)
PCB
1.688±0.013
0.067±0.005
61.173±0.008
5.598
PCB-PAC
1.717±0.012
0.074±0.004
71.318±0.001
——
PEGMA
1.121±0.041
0.445±0.012
28.898±0.006
2.264
PEGMA-PAC
1.174±0.052
0.526±0.017
31.280±0.005
——
PHEMA
0.789±0.032
0.264±0.011
——
1.943
PHEMA-PAC
0.831±0.044
0.358±0.015
——
——
Table 2 Kinetic constants for pseudo-first model (K1 min-1), pseudo-second order model (K2 g mg-1 min-1; qe mg/g) and intraparticle diffusion model (K3 mg g-1 min1/2) for adsorption of MB by PAC and PCB-PAC. Pseudo-1st-order Adsorbents
K1
R12
PAC
0.1811
0.2951
PCB-PAC
0.2354
0.6569
Pseudo-2nd-order
Intraparticle diffusion
qe.exp
qe.cal
R22
3.6101
99.49
100.00
0.9997
0.2963
95.202
0.9992
0.3537
99.19
103.09
0.9984
3.4322
39.643
0.9923
K2×10
3
K3
C
Table 3 Langmuir isotherm model (q m mg/g; KL L/mg) and Freundlich isotherm model (KF (mg/g)(L/mg) 1/n) constants and correlation coefficients for the adsorption of MB by PAC and PCB-PAC. Adsorbents PAC PCB-PAC
Fitting method
Langmuir
Freundlich 2
qm
KL
R
KF
1/n
R2
Linear Non-linear
366.3004
0.8273
0.7463
213.0591
0.1320
0.9715
380.5284
0.6080
0.6686
213.4364
0.1392
0.9569
Linear Non-linear
327.8689
0.07325
0.9771
119.2889
0.1863
0.9175
324.5723
0.08092
0.9722
124.8735
0.1810
0.9068
R32