Elsevier

Biosensors and Bioelectronics

Volume 24, Issue 4, 1 December 2008, Pages 748-755
Biosensors and Bioelectronics

Molecularly imprinted poly(ethylene-co-vinyl alcohol) membranes for the specific recognition of phospholipids

https://doi.org/10.1016/j.bios.2008.06.050Get rights and content

Abstract

In this paper we concentrated on the possibility of adopting molecular imprinting technology for the preparation of polymeric membranes imprinted with phosphatidylcholine, one of the main phospholipids found in the cell membrane and lipoproteins, via phase inversion, with the intention of applying this method in the ongoing research into the regression of atherosclerosis. The polymer matrix was based on poly(ethylene-co-vinyl alcohol) with an ethylene molar content of 44% and the amount of template molecule was varied so as to obtain three different kinds of membrane. We found that they possessed elevated binding capabilities (78.6% of the initial amount of phosphatidylcholine was found to be adsorbed by the membrane) united with a very high selectivity. Similar phospholipids (phosphatidylinositol and phosphatidylethanolamine) were found to be adsorbed only in very small quantities and mostly due to the porosity of the membrane itself and not to molecular imprinting.

Introduction

Molecular imprinting technology (MIT) allows the preparation of polymeric materials capable of selective molecular recognition ; (Wulff et al., 1986; Mosbach, 1994, Andersson et al., 1996, Shea, 1994, Wulff, 1995, Mosbach and Ramström, 1996, Steinke et al., 1995, Ye et al., 2001, Allender et al., 2000, Ciardelli et al., 2005). This means that the polymeric matrix is able to distinguish between chemical species and to bind those that exhibit certain functional groups. The high selectivity that can be obtained for these predetermined species is due to recognition sites inserted within the material through polymerisation of a monomer in the presence of the template or through phase inversion of a homogeneous polymer solution containing the molecule to be recognised (Komiyama et al., 2003, Silvestri et al., 2004). Under defined conditions the template molecule can be removed, leaving behind a cavity, complementary in shape and functionality, which will bind molecules identical to the template. The selective memory of molecularly imprinted polymers (MIPs) has been proved to be highly resistant to time and use (Komiyama et al., 2003). They are used in the construction of membrane materials for the achievement of selective diffusion and bio-separation, as chromatographic supports, to provide the recognition element for chemical sensors, and for the synthesis of polymeric materials that mimic biological receptors (Silvestri et al., 2004, Silvestri et al., 2005a, Silvestri et al., 2005b, Ciardelli et al., 2004).

One of the aims in this field is to find innovative biomedical uses for extra corporeal purification systems or implantable devices capable of recognition and also the preparation of materials exhibiting molecular recognition towards biological compounds for application in tissue engineering (Ciardelli et al., 2005). Polymer membranes for clinical use must therefore be highly biocompatible and possess a specific permeability and bio stability.

In this paper we deal with the preparation of MIPs where the polymer matrix is a membrane obtained by phase inversion (Silvestri et al., 2006, Ulbricht, 2004). With this method there are two principal components: a homogeneous polymer solution and a non-solvent. The polymer solution is transformed in a two-phase system where the polymer-rich phase forms the final solid membrane structure, and the polymer-lean phase forms the membrane pores through the exchange of solvent and non-solvent across the interface. After imprinting there are two important stages known as template extraction and rebinding. Both of them must leave the morphology of the matrix unaltered and are necessary for recognition and selectivity tests.

In this paper we focused particularly on the possibility of imprinting phosphatidylcholine, one of the main phospholipids found in the cell membrane and lipoproteins, in a poly(ethylene-co-vinyl alcohol) membrane for potential use in extra corporeal purification systems, such as dialysis or plasmapheresis. The polymer in question is thermoplastic and highly haemocompatible. It has often been studied for the creation of porous membranes (Young et al., 1999a, Young et al., 1999b, Young et al., 2000), and previous studies of ours have shown promising results as a molecularly imprinted material in the form of membranes (Silvestri et al., 2004, Silvestri et al., 2005a, Silvestri et al., 2005b, Silvestri et al., 2006).

Cholesterol is a molecule found in the cell membranes of all body tissues and is transported in the blood flow via lipoproteins. There are different kinds of lipoproteins, the two most relevant being low density lipoprotein (LDL) high density lipoprotein (HDL) (Otto, 2005). LDL carries cholesterol towards peripheral tissues while HDL carries it from the peripheral tissues towards the liver, where it will be eliminated. Cholesterol is involved in many biochemical processes and is required to build and maintain cell membrane structure. It aids in the manufacture of bile and is the major precursor for the synthesis of vitamin D and various hormones. It is also associated with many cardiovascular diseases, such as atherosclerosis, which affects the arterial blood vessel, and is caused by the formation of multiple plaques due to elevated concentrations of LDL cholesterol and low concentrations of HDL cholesterol. These plaques cause the formation of blood clots that will rapidly slow or stop the blood flow, leading to death of the tissues fed by the artery.

A reduction in blood levels of LDL cholesterol should result in a regression of atherosclerosis, for this purpose we studied the possibility of creating a system capable of recognising and separating these molecules. An interesting way to reduce the level of cholesterol or other lipids that are etiological in atherosclerosis could be the use of an extra corporeal device containing a selective adsorbent prepared for example by molecular imprinting technique.

Molecular imprinted polymers are being investigated more and more as selective adsorbents for cholesterol (Asanuma et al., 2000, Perez et al., 2001, Kugimiya et al., 2001, Davidson et al., 2003). Whitcombe et al. (1995) prepared cholesterol selective adsorbents by a covalent molecular imprinting while Sellergren et al. (1998) synthesized polymerisable derivatives of cholesterol and bile acids to be used as amphiphilic monomers in the imprinting of highly cross-linked methacrylates with cholesterol. Hwang and Lee (2002) used cholesteryl (4-vinyl) phenyl carbonate for a covalent imprinting, linker. Gore et al. (2004) prepared cholesterol conjugated monomers and hydrophilic cross-linkers and in addition materials showing cross-linkers containing covalently linked cholesterol, both systems showing recognition capacity in water.

Section snippets

Materials

Poly(ethylene-co-vinyl alcohol) (EVAL, Sigma–Aldrich, USA, intrinsic viscosity 8.44 dL/g at 35 °C), with an ethylene molar content of 44%, was used as the biocompatible polymer matrix. Solvents used were dimethyl sulphoxide (DMSO, Riedel-de Haën, Germany) and tetrahydrofuran (THF, Carlo Erba Reagenti, Italy). Phosphatidylcholine (PC, Sigma–Aldrich, USA), phosphatidylethanolamine (PE, Sigma–Aldrich, USA) and phosphatidylinositol (PI, Sigma–Aldrich, USA) were the three phospholipids used as

Morphologic, chemical and physical characterization

The thermal investigation (DSC and TGA) and spectroscopic FT-IR analysis, that are normally utilized for the evaluation of typology and number of interactions between the components of a polymeric blend, in the field of molecular imprinting could be useful for the preliminary determination of template–MIP interaction degree: this knowledge may be very important for the prediction and confirmation of the stability and interaction capacity of recognition cavities towards template molecule and

Conclusions

In the present work it was possible to obtain and characterise an imprinted polymeric membrane capable of recognising phospholipids.

The poly(ethylene-co-vinyl alcohol) membranes were created via phase inversion in the presence of the template molecule, in this case phosphatidylcholine. These membranes contained different amounts of PC and for every one of them we determined the ability to release and to selectively recognise the molecule. The MIMs thus obtained were put within a permeability

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