Supermacroporous poly(hydroxyethyl methacrylate) based cryogel with embedded bilirubin imprinted particles
Introduction
Molecular imprinting is a technology to create recognition sites in a macromolecular matrix using a molecular template [1]. In other words, both the shape image of the target and alignment of the functional moieties to interact with those in the target, are memorized in the macromolecular matrix for the recognition or separation of the target during formation of the polymeric materials themselves [2]. Molecular imprinting has been used successfully for imprinting of small molecules, metal-ions and proteins [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15]. Due to the small number of publications, quantitative experimental data on bilirubin-imprinted polymers is very limited. Molecularly imprinted polymers can be stable even in critical and physical conditions, have a life of several years without any apparent reduction in performance and can be used repeatedly without any alteration to the memory [16]. These materials are cheap to synthesize and can be manufactured in large quantities with good reproducibility. Therefore, MIPs can be considered as an artificial affinity media. Molecular recognition-based separation techniques have received much attention in various fields because of their high selectivity for target molecules [17].
Bilirubin is an important bioactive molecule which is produced from the hemoglobin metabolism. It is transported to the liver as a complex with albumin where it is normally conjugated and excreted into the bile [18]. The free bilirubin is toxic. High concentrations of free bilirubin can evoke hepatic or biliary tract dysfunction and permanent brain damage or death in more severe case [19]. Neurological dysfunctions as kernicterus or bilirubin encephalopathy may develop if the bilirubin concentration in the plasma rises above 15 mg/dL. Disorders in the metabolism of bilirubin may cause a yellow discoloration of the skin and other tissues.
Several techniques have been developed for the treatment of hyperbilirubinemia such as phototherapy, plasma exchange, hemodialysis and hemoperfusion [20], [21], [22], [23], [24], [25], [26], [27]. In recent years, molecular imprinted polymers have developed into a powerful tool for the specific recognition of toxic substances. Different kinds of MIP particles are manufactured for bilirubin recognition [28], [29], [30], [31]. But, there is only one study that uses MIP particles for selective removal of bilirubin from human plasma [32]. We report herein our experience on the use of selective bilirubin removal with poly(hydroxyethyl methacrylate) cryogel with embedded bilirubin-imprinted poly(hydroxyethyl methacrylate-N-methacryloyl-(l)-tyrosine methylester) particles [PHEMA/MIP composite cryogel]. Cryogels are a very good alternative to bioseparation with many advantages including large pores, short diffusion path, low pressure drop and very short residence time [33], [34], [35]. But, due to the existing of large pores within the cryogel, the adsorption capacity for the biomolecules is low [36]. In actual bioseparation processes, it is of great importance to improve the binding capacity of supermacroporous cryogel. Therefore, particle embedding would be a useful improvement mode to use in the preparation of novel composite cryogels for increasing surface area [37], [38], [39]. This approach makes use of a combinatorial selection strategy to enhance adsorption capacity. Bilirubin adsorption and selectivity studies versus other competitive substances such as cholesterol and testosterone are reported here. Finally, repeated use of the PHEMA/MIP composite cryogel has been also studied as well.
Section snippets
Materials
Bilirubin, cholesterol, testosterone, l-tyrosine methylester, methacryloyl chloride, were purchased from Sigma (St. Louis, USA). Hydroxyethyl methacrylate and ethylene glycol dimethacrylate (EGDMA) were obtained from Sigma (St. Louis, USA), distilled under reduced pressure in the presence of hydroquinone inhibitor and stored at 4 °C until use. Ammonium persulfate (APS) and N,N,N′,N′-tetramethylene diamine (TEMED) were obtained also from Sigma. Methanol was HPLC grade and were supplied from Sigma
Characterization of cryogel
The SEM images of the internal structures of the PHEMA cryogel and PHEMA/MIP composite cryogel are shown in Fig. 1. PHEMA/MIP composite cryogel produced in such a way have porous and thin polymer walls, large continuous interconnected pores (10–100 μm in diameter) that provide channels for the mobile phase to flow through. SEM images showed that the MIP particles were uniformly distributed into the PHEMA cryogel network. Pore size of the matrix is much larger than the size of the bilirubin
Conclusion
The results presented here demonstrate that the PHEMA cryogels embedded with MIP particles can be used for the recognition and selective removal of bilirubin molecules from human plasma. This recognition may be through a multistep binding, with the specificity conferred by hydrophobic interactions and shape selectivity. Because, the MIP particles are located, or close to, the macropore surface, these MIP particles have a good site accessibility toward, the target bilirubin molecules in human
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