Synthesis and characterization of MIPs - a viable commercial venture

Knowledge and applications of molecularly imprinted polymers (MIP), is rapidly developing, especially in the chemical analysis field, Molecular imprinting is a method of inducing molecular recognition properties in synthetic polymers in response to the presence of a template species during formation of the three-dimensional structure of the polymer.

Accordingly, the projected US market for separation techniques will be approximately $1.19 billion. MIP materials could claim a share of 1–3% of this market particularly in the sector of chromatography columns, which is valued at $500 million per annum. The ability of imprinted polymeric materials to resolve racemic mixtures could prove especially lucrative to the pharmaceutical industry where as much as 68% of drugs currently in development are chiral. This corresponds to a market for chiral compounds of $120 billion. The broad selection of MIP applications already within reach for commercial applications ranging from separations, assays, and sensors to advanced catalysis will be complemented in future by advanced uses such as detection of gases in space exploration and drug-release matrices Molecularly imprinted polymers can be a valuable alternative for receptors of biological origin, such as antibodies, in terms of robustness, versatility and ease of preparation. The synthesis, characteristic, effect of molecular recognition and different preparation methods of MIP in recent few years are discussed in this review, unsolved problems and possible developments of MIP were also been briefly discussed.

Introduction

Drug delivery systems are required whenever an administered therapeutic agent needs to be protected against metabolic attack, or when there are absorption barriers or dosage limitations. The ideal delivery vehicle will ensure that the drug is released at the right site, in the right dose and for the required time. It will also be biocompatible or biodegradable such that the delivery system is transformed into non-toxic fragments that are eliminated harmlessly from the body. The importance of this field of research is growing as ever more complex drugs and biopharmaceuticals are being developed, many of which cannot be administered without a controlled dosage system. Polymeric materials have been used for some time as drug delivery systems, most widely as implantable materials. Delayed release devices of this type have the advantage of increasing the residence time of a drug within a patient, ensuring better compliance with most dosage requirements, or in the case of those compounds that have a narrow therapeutic window, maintaining the concentration below levels where potential harmful side effects become prevalent. The simplest polymeric delivery systems are those where the drug is dispersed randomly within the polymer matrix and released as a consequence of erosion of the carrier in vivo. Although simple to prepare, these materials have the disadvantage that the drug can be released suddenly through breakdown of the matrix past a critical threshold, leading to potentially serious consequences for the patient if the drug is harmful in high concentrations. However, there are still a number of problems with many polymeric drug delivery systems that have limited their practical application. A particular issue is to effect feedback-controlled release, i.e. the maintenance of a therapeutic level of a drug within both the drug reservoir and the target site. This requires a drug delivery system with molecular recognition properties, such that it is able to bind and release only very specific molecular species under conditions where equilibrium concentrations may be critical. Molecularly imprinted polymers (MIPs) have been a focus of research as a consequence of their molecular recognition properties combined with facile synthesis and, as we aim to show in this short review, are now receiving considerable attention for drug delivery applications.

Molecular recognition is defined as ‘‘the ability of one molecule to attach to another molecule that has a complementary shape.  Molecular imprinting technology can provide efficient polymer systems with the ability to identify specific bioactive molecules and a sorption capacity dependent on the properties and template concentration of the surrounding medium; therefore, although imprinted DDS have not reached clinical application yet, this technology has an enormous potential for creating satisfactory dosage forms. A good proof of this is the progressive increase in the number of papers devoted to the application of MIPs in the design of new DDS. The devices useful in closely related fields, such as diagnostic sensors or chemical traps to remove undesirable substances from the body, Enormous interest has also been shown in imprinted materials as they act out biological receptors for the screening of new substances with potential pharmacological activity or to specifically detect drugs in biological fluids in screening assays for drugs of abuse. Such specificity is comparable with monoclonal antibodies used in immunoassay techniques.

This study aims to present the principal synthetic considerations pertaining to good practice in the polymerization aspects of molecularly imprinted polymers (MIP), It is also aimed for researchers to get familiar with MIP methods. It is our hope that this will facilitate researchers to plan their own syntheses of MIP in a more logical and structured fashion, as well as the scope for rationally designing improved imprinted materials in the future. The challenge of designing and synthesizing a MIP can be a daunting prospect to the uninitiated practitioner not merely because of the sheer number of experimental variables involved,

A Brief History Of Imprinting

Molecular imprinting is not a new science. The earliest reports of imprinting go back to the early 1930s when a Soviet chemist M.V. Polyakov ^[1] prepared a number of silica gels and observed that when prepared in the presence of a solvent additive the resulting silica demonstrated preferential binding capacity for that solvent. A later study, also using silica, was to have more of an impact. In 1949 a senior student of Linus Pauling, Frank Dickey, published the results of experiments where silica gels had been prepared in the presence of dyes^[2]. Dickey observed that after removal of the ‘patterning’ dye the silica would rebind the same dye in preference to the others. Silica imprinting continued during the 1950s and 1960s but the number of publications in the area remained low. However, in 1972 a step change in molecular imprinting occurred when the group of Guenter Wulff reported that they had successfully prepared a molecularly imprinted organic polymer ^[3]. Wulff used what is now termed as ‘covalent approach’ to prepare an organic molecularly imprinted polymer capable of discriminating between the enantiomers of glyceric acid. Subsequently, throughout the 1970s and 1980s, Wulff’s group published extensively using this approach. The second major break through in organic polymer imprinting occurred in 1981 when Mosbach and Arshady reported that they had prepared an organic MIP using non-covalent interaction only ^[4]. This approach was termed the dnon-covalent approach T as opposed to the covalent approach favored by Wulff, and it was this approach, with its simple, seemingly trivial methodology, that triggered the explosion in molecular imprinting that was to occur during the 1990s. To this day the non-covalent versus covalent debate continues with both sides being championed. However, it is generally accepted that there are pros and cons to both approaches and in 1995 Whitcombe et al. reported an intermediate approach that appeared to combine the advantages of both approaches ^[5]. This approach relies on covalent interaction during the polymerization stage but non-covalent interactions during rebinding. Importantly, in order to improve subsequent non-covalent binding geometry, Whitcombe’s approach incorporated a sacrificial spacer group that was designed to be lost during template removal. The non-covalent approach however is still by far the most widely used approach in MIP synthesis. Several of its drawbacks can be overcome by the use of stoichiometrically associating monomer-template systems ^{[6, 7, 8, 9]}. This has resulted in a range of receptors exhibiting high capacity and effective recognition properties in aqueous media.

Rationale for imprinted polymer drug delivery systems

Molecularly imprinted polymers have already found many uses in analytical chemistry, separation science, as catalysts and artificial enzymes. The synthesis of these materials and their general properties are covered extensively elsewhere ^[10], but for biomedical applications there are a number of specialized requirements relating to imprinted polymer preparation. Most of the molecularly imprinted polymers rely upon a high degree of cross-linking in order to fix the spatial orientation of functional groups, which is generally thought to be required for the imprinted material to retain its molecular recognition capabilities ^{[11, 12]}. However, for use in drug delivery, it is usually advantageous to prepare molecularly imprinted polymer gels that are not quite as densely cross-linked. This is especially true where the polymers are synthesized with water-soluble monomers as many hydrogels may display a change in their swelling behavior under the influence of an external stimulus ^[13]. This external stimulus can be due to a change in temperature, pH, ionic strength, solvent quality, presence or absence of chemical species, electric fields and irradiation with UV or visible light. This change in the degree of swelling can be used to modulate the capture and release of the imprinted template molecule, as well as to improve the separation capabilities of imprinted polymers when used as ‘clean-up’ or scavenging devices. Imprinted materials are also appealing as drug delivery vehicles because, owing to their cross-linked polymeric nature, they inherently act as reservoirs for low molecular weight species. They can potentially increase the residence time of drug within the body by reducing the rate at which the drug is released. In cases where the drug has a narrow therapeutic window, MIP delivery vehicles might keep the concentration of drug in body below the concentration where adverse side effects become dominant. The use of imprinted hydrogels for drug delivery has an added advantage as a change from a collapsed to a swollen state can be chosen to suit the conditions where the polymer is going to be used to release its load of therapeutic agent. This can be envisaged in terms of an imprinted hydrogels that collapses to protect its therapeutic payload through the GI tract but expands to release the drug in the small intestine or the colon. The modulation of drug release from an imprinted polymer by a feedback control mechanism can also be considered, such as insulin release when blood glucose level rises above a minimum threshold level, or release of a drug when the imprinted polymer encounters a specific target agent, such as a protein or cell surface receptor. One further potential advantage of imprinted polymers as drug delivery devices is that, in the case where a racemic mixture of a drug is used, they can selectively release the more effective enantiomer. ^{[14, 15, 16].}

Polymer synthesis

Free radical polymerization

Free radical (or chain growth) polymerization is the most important synthetic method available today for the conversion of monomer into polymer. Numerous vinyl monomers can be polymerised very effectively in excellent yields by free radical polymerisation methods, including ethylene, styrene and methyl methacrylate which are of particular industrial importance. Free radical polymerizations can be performed under mild reaction conditions (e.g. ambient temperatures and atmospheric pressures) in bulk or in solution, and are very tolerant of functional groups in the monomers and impurities in the system (e.g. water). It is for these reasons, as well as the fact that many vinyl monomers are available commercially at low cost, that free radical polymerisation is usually the method of choice for preparing molecularly imprinted polymers. The mechanism of free radical polymerisation is characterized by three distinct stages: (1) initiation, (2) propagation, and (3) termination. But it is worthwhile emphasizing two points. First of all, in a typical free radical polymerisation the rate of propagation (chain growth) is usually much faster than the rate of initiation, such that as soon as a new polymer chain starts to grow it propagates to high molecular weight in a relatively short period of time (perhaps within a second or two) before it terminates. What this means is that high molecular weight product is present in the system even when the amount of monomer consumed is low. Second of all, the source of free radicals (the initiator) is normally active over the entire duration of the polymerisation, one would observe the presence of un-reacted monomer and initiator, propagating (growing) polymer chains and high molecular weight polymer chains that were terminated (dead). The rate and mode of decomposition of an initiator to radicals can be triggered and controlled in a number of ways, including heat, light and by chemical/electrochemical means, depending upon its chemical nature. For example, the azo initiator azobisisobutyronitrile (AIBN) can be conveniently decomposed by photolysis (UV) or thermolysis to give stabilised, carbon-centred radicals capable of initiating the growth of a number of vinyl monomers. As an illustrative example of the use of AIBN, or indeed other initiators, to polymerise vinyl monomers, AIBN can polymerise methyl methacrylate under thermal or photochemical conditions to give poly(methyl methacrylate) (Fig. 1), i.e. PerspexTM, a linear macromolecule that would be soluble in a thermodynamically compatible solvent such as toluene or tetrahydrofuran.

Fig. 1 conversion of methyl methacrylate monomer by free radical polymerization into poly(mehyl methacrylate)

 Free radical copolymerization

It is often highly desirable, not only in molecular imprinting circles, to simultaneously polymerise (copolymerise) two or more vinyl monomers within the same reaction vessel to give copolymers (as opposed to a homopolymer, which arises from the polymerization of one single monomer). This allows products to be prepared with chemical properties distinct to the polymers obtained upon polymerizing each monomer independently. For example, methyl methacrylate could be copolymerised with the more hydrophobic monomer butyl methacrylate to yield a copolymer product where, for any given polymer chain, there would be a statistical distribution of methyl methacrylate and butyl methacrylate units along the length of the polymer chain, and where the statistical distribution would be dependent upon the relative concentrations of the two monomers in the feed prior to polymerisation (Fig. 2a). The linear copolymer product, poly (methyl methacrylate-co-butyl methacrylate), would be soluble in a thermodynamically compatible solvent. Particular care must be exercised in free radical copolymerisations to take account of the relative reactivities of the constituent monomers and to appreciate that all monomers are not consumed at the same rate, else the chemical composition of the copolymer products and the distribution of the monomer units within the copolymers may well be dramatically different to what one would predict on the basis of the monomer feed composition alone. As simple illustrative examples of this idea, certain pairs of monomers copolymerize to give specifically alternating copolymers (e.g. stilbene and maleic anhydride, Fig. 2b) irrespective of the monomer feed composition, whereas other pairs of monomers (e.g. maleimide and maleic anhydride) copolymerize inefficiently or not at all. It must also be pointed out that for any given pair of comonomers, the molecular composition of the resultant copolymer and the distribution of the monomer units within the copolymer are also dependent upon the relative monomer feed concentrations, and that this can vary with time. Fortunately, the relative reactivities of many common monomers are known and have been tabulated, normally in the form of reactivity ratios for given pairs of monomers ^[17,18,19].

Fig 2. Free radical copolymerization of; (a) methyl methacrylate with n-butyl methacrylate, and (b) stilibene and maleic anhydride polymer (a) is a random copolymer whereas polymer (b) is a specially altering copolymer

Cross-linked polymers

All the polymerisations discussed hitherto involve the propagation (growth) of polymers derived from monomers with one single polymerisable vinyl group, hereafter referred to as mono-functional monomers. Mono-functional monomers normally polymerise to give linear macromolecules that are soluble in chemically compatible solvents. When multi-functional monomers, i.e. monomers bearing two or more polymerisable vinyl groups, are polymerised, either on their own or in combination with a comonomer or comonomers, then the outcome is quite different and this allows a number of non-linear polymer architectures of high commercial value to be prepared. These materials may be soluble or insoluble, and can be conveniently classified as branched macromolecules, microgels and macroscopic networks (Fig. 3) ^[19].

Fig.3. Schematic representation showing polymers with different topologies: linear,  branched macroscopic, network and microgel    

Multi-functional monomers are more commonly referred to as cross-linkers, and serve to chemically link two or more linear polymer chains To quote but one technologically relevant example, Merrifield resin ^[20,21] has found application in catalysis and solid-phase organic chemistry and can be readily prepared by the copolymerisation of styrene (as the mono-functional monomer) with divinylbenzene (as the cross-linker) to give a poly(styrene-co-divinylbenzene) macroscopic network, the chemical structure of which can be elaborated further if so desired (Fig. 4).

Fig. 4. Schematic representation of the cross-linked polymer network arising from the Copolymerization of styrene with p-divinylbenzene

In the world of molecular imprinting, macroscopic polymer networks have been the non-linear polymers most widely synthesized and studied, as these tend to be insoluble species that lend rigidity and impart mechanical stability to an imprinted binding site. There have been some reports in the literature describing the imprinting of (soluble) microgels and linear macromolecules, but these are relatively few in number. For these reasons It will be focused exclusively hereafter upon the synthesis of insoluble macroscopic network polymers, concentrating upon their distinct physical properties and illustrating how these properties can be dramatically influenced by tailoring of the conditions under which they are prepared.

MIP syntheses

The basic strategy

Molecularly imprinted polymers are crosslinked polymeric materials that exhibit high binding capacity and selectivity against a target molecule (template) purposely present during the synthesis process. The concept behind the selective binding site formation is schematically shown in Fig. 5. In solution, the template interacts with functional monomers or precursors and the structure of these aggregates or complexes is preserved by copolymerisation in the presence of an excess amount of a crosslinker. After polymerisation, the template is removed from the polymer matrix, thus leaving internal images with specific binding sites in the material.

Fig. 5 schematic representation of the imprinting process

The challenge of designing and synthesizing a molecularly imprinted polymer (MIP) can be a daunting prospect to the uninitiated practitioner, not least because of the sheer number of experimental variables involved, e.g. the nature and levels of template, functional monomer(s), cross-linker(s), solvent(s) and initiator, the method of initiation and the duration of polymerisation. Fortunately, a good number of “rules of thumb” have emerged in a number of factors pertaining to the template molecule and the selection of suitable functional monomers, cross-linkers, solvents, initiators and general polymerisation procedures.

Template

In all molecular imprinting processes the template is of central importance in that it directs the organisation of the functional groups pendent to the functional monomers. Unfortunately, and for a variety of reasons, not all templates are directly amenable to templating. In terms of compatibility with free radical polymerisation, templates should ideally be chemically inert under the polymerisation conditions, thus alternative imprinting strategies may have to be sought if the template can participate in radical reactions or is for any other reason unstable under the polymerisation conditions. The following are legitimate questions to ask of a template: (1) Does the template bear any polymerisable groups, (2) Does the template bear functionality that could potentially inhibit or retard a free radical polymerisation, e.g. a thiol group or a hydroquinone moiety, and (3) Will the template be stable at moderately elevated temperatures (e.g. at or around 60C if AIBN is being used as the chemical initiator) or upon exposure to UV irradiation.

Fig.6. Structures of templates or analogues

Functional monomers

Functional monomers are responsible for the binding interactions in the imprinted binding sites and, for non-covalent molecular imprinting protocols, are normally used in excess relative to the number of moles of template to favor the formation of template, functional monomer assemblies (template to functional monomer ratios of 1:4 and upwards are rather common for non-covalent imprinting). It is clearly very important to match the functionality of the template with the functionality of the functional monomer in a complementary fashion (e.g. H-bond donor with H-bond acceptor) in order to maximise complex formation and thus the imprinting effect. When two or more functional monomers are used simultaneously in “cocktail” polymerisation ^[22] it is however also important to bear in mind the reactivity ratios of the monomers to ensure that copolymerisation is feasible (see earlier). In passing, it is also worth noting that complexation of a template by a functional monomer can also influence the reactivity of the monomer to some extent, as a result of pertubations to the electronics and/or the sterics of the monomer. Scores of functional monomers with chemically diverse structures and polarities are commercially available and many more can be prepared by rational design. In Fig. 7 the chemical structures of a selection of the more important functional monomers are shown.

Fig.7 - Selection of monomers used in the non-covalent approach. Acidic; aI: methacrylic acid (MAA); aII: p-vinylbenzoic acid; aIII: acrylic acid (AA); aIV: itaconic acid; aV: 2-(trifluoromethyl)-acrylic acid; aVI: acrylamido-(2-methyl)-propane sulfonic acid (AMPSA). Basic; bI: 4-vinylpyridine ; bII: 2-vinylpyridine (2-VP); bIII: 4-(5)-vinylimidazole; bIV: 1-vinylimidazole; bV: allylamine; bVI: N,N _-diethyl aminoethyl methacrylamide, bVII: N-(2-aminethyl)-methacrylamide; bVIII: N,N _ diethyl-4-styrylamidine; bIX: N,N,N,-trimethyl aminoethylmethacrylate; bX: N vinylpyrrolidone; bXI: urocanic ethyl ester. Neutral; nI: acrylamide; nII: methacrylamide; nIII: 2-hydroxyethyl methacrylate nIV: trans-3-(3-pyridyl)-acrylic acid; nV: acrylonitrile (AN); nVI: methyl methacrylate (MMA); nVII: styrene; nVIII: ethylstyrene.

Cross-linkers:-

In an imprinted polymer the cross-linker fulfils three major functions.

(1) The cross-linker is important in controlling the morphology of the polymer matrix,  whether it is gel-type, macroporous or a microgel powder.

(2)  It serves to stabilise the imprinted binding site.

(3) It imparts mechanical stability to the polymer matrix.

Much has been written about the effect of the cross-linker on the molecular recognition behaviour of imprinted polymers, but from a polymerisation point of view, high cross-link ratios are generally preferred in order to access permanently porous (macroporous) materials and in order to be able to generate materials with adequate mechanical stability. Polymers with cross-link ratios in excess of 80% are oftenly used. For the same reason that one should match the reactivity ratios of functional monomers in a cocktail polymerization to ensure smooth incorporation of the comonomers, the reactivity ratio of the cross-linker should ideally also be matched to that of the functional monomer(s).. It should also be borne in mind that there may well be chemically distinct vinyl groups in multi-functional monomers with distinct reactivity ratios, i.e. different vinyl groups may be incorporated at differential rates into the polymer. Quite a number of cross-linkers compatible with molecular imprinting are known, many of which are commercially available and a few of which are capable of simultaneously complexing with the template and thus acting as functional monomers. The chemical structures of several well-known cross-linkers are shown in Fig. 8.

Figure8. Selection of cross-linkers used for molecular imprinting

Solvents (Porogens)

The solvent serves to bring all the components in the polymerisation, i.e. template, functional monomer(s), cross-linker and initiator into one phase. However, it serves a second important function in that it is also responsible for creating the pores in macroporous polymers. For this reason it is quite common to refer to the solvent as the “porogen”. When macroporous polymers are being prepared, the nature and the level of the porogen can be used to control the morphology and the total pore volume. More specifically, use of a thermodynamically good solvent tends to lead to polymers with well developed pore structures and high specific surface areas, use of a thermodynamically poor solvent leads to polymers with poorly developed pore structures and low specific surface areas. Increasing the volume of porogen increases the pore volume. Besides its dual roles as a solvent and as a pore forming agent, the solvent in a non-covalent imprinting polymerisation must also be judiciously chosen such that it simultaneously maximises the likelihood of template, functional monomer complex formation. Normally, this implies that apolar, non-protic solvents, e.g. toluene, are preferred as such solvents stabilise hydrogen bonds, however if hydrophobic forces are being used to drive the complexation then water could well be the solvent of choice.

Initiators

In principle, any of the methods of initiation described earlier can be used to initiate free radical polymerisations in the presence of templates. However, there may well be drivers for selecting one over another arising from the system under study.  For example, if the template were photochemically or thermally unstable then intiators that can be triggered photochemically and thermally, respectively, would not be attractive. Where complexation is driven by hydrogen bonding then lower polymerisation temperatures are preferred, and under such circumstances photochemically active initiators may well be preferred as these can operate efficiently at low temperature. The chemical structures of selected polymerisation intiators are shown in Fig. 9.

Fig 9: Chemical structures of selected chemical initiators

Category of MIP

Essentially, two kinds of molecular imprinting strategies have been established based on covalent bonds or non-covalent interactions between the template and functional monomers (Figure 1). In both cases, the functional monomers, chosen so as to allow interactions with the functional groups of the imprinted molecule, are polymerized in the presence of the imprinted molecule. The special binding sites are formed by covalent or, more commonly, non-covalent interaction between the functional group of imprint template and the monomer, followed by a crosslinked co-polymerization [23]. Of the two

strategies, the non-covalent approach has been used more extensively due to following three reasons:

(1)Non-covalent protocol is easily conducted, avoiding the tedious synthesis of prepolymerization complex.

(2) Removal of the template is generally much easier, usually accomplished by continuous extraction.

(3) A greater variety of functionality can be introduced into the MIP binding site using non-covalent methods.

Fig. 10.Schematic representation of covalent and non-covalent molecular imprinting procedures

Epilogue

Because optimization of MIP formulations is largely empirical, high-throughput methods have recently been introduced for rapid screening of the different parameters ^{[23,24,25,26]}. These methods make use of semi-automated procedures for parallel synthesis of small quantities of MIPs with different compositions. Rapid assays can be conducted to quickly determine candidates with improved binding or selectivity properties; these candidates can then be scaled up for more detailed analysis. Furthermore, chemometric tools for multi-variate analysis of screened libraries are also being developed to aid the optimization process [^27,28,29,30]. These methods have been used to optimize X /M ratios in MIPs, as well as T /M ratios, type and amount of porogen, and initiation variables. In addition to these experimental methods, computational approaches are being developed for ‘virtual’ high-throughput and combinatorial searches for functional group mixtures that can provide insight toward optimal MIP formulations. A last consideration is that all optimization methods discussed here apply to traditional formation of MIPs in bulk polymer materials; different formats such as surface imprinting, small particle MIPs, and imprinting in thin films may have additional or different rules guiding their optimization. Finally, applications in the field of drug delivery may entertain alternative optimization variables such as mass transfer rates, control of swelling properties, and bioavailability.

Spectroscopic evaluation of template–monomer interactions

NMR

The direct evidence for the formation of non-covalent monomer–template interactions, the extent of which is reflected in the total binding term, ΔΔG _bind, was first presented in an NMR study by Sellergren et al. ^[31]. The results suggested the minor presence of template self-association and higher order complexes. Whitcombe et al. also adopted a similar approach, where NMR chemical shift studies allowed the calculation of dissociation constants and a potential means for predicting the binding capacities of MIPs ^[32]. The NMR characterizations of functional monomer–template interactions have also been applied to the study of the interactions between many other functional monomers and templates ^[33–54]. In most of these studies, it was also possible to determine the exact composition of the complex. For example, Nicholls investigated in detail the characterization of complexes between nicotine and methacrylic acid ^[52]

 FTIR

Fourier transform infrared spectroscopy (FTIR) is a technique capable to determine the modification of the structure of a molecule. It has been used particularly with samples in solution or in the solid state. The imprinting process begins with a complexation between a functional monomer and a template generally via hydrogen bonding. The formation of this bond can be readily identifiable using FTIR since the stretching frequency of hydroxyl or amino groups (hydrogen bond donors) and carbonyl groups (hydrogen bond acceptors) are displaced and an observable shift can be identified. However, the presence of solvent can interfere on the determination of specific characteristics of complexation. This explains why this technique is not well applied to the pre-polymerisation solution analysis ^[55,56]. Brune et al. investigated the interaction by FTIR between phenolic compounds and an analogue of the acrylic ester monomer, ethyl propionate in hexane ^[57}.  Duffy et al. characterised by FTIR pre- and postpolymerisation binding properties of an imprinted membrane for thymine using 2,6-diacyldiaminopyridine as a functional monomer ^[56]. Liquid spectroscopy clearly shows the three hydrogen bonds linking the monomer and template molecules in the pre polymerization mixture. The three characteristic bands of the amino groups above 3000 cm^-1 remained after the polymerisation, indicating that the template– monomer complex was still present in the polymer. However, the binding constant of the imprinted membrane for the thymine was estimated to be ten times lower that that of the soluble monomer. This decrease was attributed to a shrinkage during cross-linking and loss of freedom of the monomer.

UV

Complexation has also been studied using UV spectroscopic titrations in order to calculate the dissociation constants for the solution adducts and the relative concentration of fully complexed templates in the polymerisation mixture ^[58]. This approach was also used to verify the inert nature of the cross-linking agent ethylene glycol dimethacrylate and for the screening of candidate functional monomers ^[59]. In another paper, Striegler and Tewes used UV spectroscopy to choose the best ligand for copper capable of providing effective functional monomers for carbohydrate imprinted polymer synthesis ^[60]. The experimental data firstly estimated the copper–ligand stability in order to select the best ligand, and secondly to evaluate the apparent binding constants between the copper-containing functional monomers and different hexoses in alkaline pH. The complex [(diethylenetriamine) copper(II)] dinitrate appeared to be the most promising monomer for the complexation with sugars, and it was found that the binding involved only hydroxyl groups at C1 and C2 of the carbohydrates. The main advantage of this technique is its simplicity of use and the possibility to control monomers–template complex formation in aqueous media ^[60,61]. Ping et al. ^[62] synthesized an MIP from the photoinduced polymerisation of acrylamide and butylenes diacrylate in the presence of l-2-chloromandelic acid (l-2-MDA) as a template and was used as a highly selective separation material for l-2-MDA. UV analysis showed the template l-2-MDA and functional monomer acrylamide formed complexes before polymerisation. The structures of complexes were simulated by using HyperChem and it was found that the binding capacity of the MIP to l-2-MDA was higher than that of other analogues with the chiral separation factor of 1.76. Scatchard analysis suggested that the MIP recognized l-2-MDA with two classes of binding sites, which was in agreement with the two kinds of complexes simulated models using HyperChem.

Computer simulation

In principle, the broad range of functional monomers currently available makes it possible to design an MIP specific for any type of stable chemical compound. The application of previous methods to select the best monomers for polymer preparation is not trivial in practice. The problem lies in the technical difficulty of performing detailed thermodynamic calculations on multi-component systems and the amount of time and resources required for the combinatorial screening of polymers. To check a simple two-component combination of 100 monomers one has to synthesise and test more than 5000 polymers which is a very difficult task. This task will be further complicated by the possibility that these monomers could be used in monomer mixtures in different ratios. One potential solution to the problem of polymer design lies in molecular modelling and in performing thermodynamic calculations with the aid of a computer developed by Piletsky et al. ^[63–66]. At present, the molecular modelling of complex systems such as molecularly imprinted polymers, their structure and possible interactions with template, solvent and other molecules, is difficult due to the extremely large computational workload required for such complex systems. However, the authors lowered the computational requirements by simplifying the model.

Characterization

The highly crosslinked network MIP materials formed during the molecular imprinting process are part of a class of materials known as macroporous polymers ^[67,68]. MIPs are solids, and therefore cannot be characterized by more commonly employed polymer characterization methods that would require polymer solutions; e.g. gel permeation chromatography, solution NMR techniques, and UV measurements directly on the polymers. Furthermore, because MIPs are amorphous, crystallographic or microscopy methods cannot be used to determine the structure of the MIP binding sites, although microscopy has aided the macroscopic understanding of MIP morphology^[69]. Therefore, there are only a limited number of direct physical characterization methods for imprinted polymers. These include surface area and porosity measurements, IR spectroscopy, solid state NMR (13CPMAS spectroscopy), and swelling. The spectroscopy methods presented are best for investigating molecular level features of the MIP materials. The surface area, porosity, and swelling measurements characterize macroscopic features of MIPs; however, information provided by these on the binding site structure of MIPs is very limited. On the other hand, this data can be very useful for drug delivery applications such as controlling substrate release times, and swelling properties due to environmental factors.

Surface area and porosity

The morphology of MIPs, shown in fig 10, arises from nuclei that form around the initiator which grow to 10–30 nm in diameter which then aggregate to form microspheres, that aggregate themselves into larger clusters that form the body of beads. The porosity and resulting surface area in MIPs is formed from irregular voids located between clusters of the microspheres (macropores, N50 nm in diameter), or from the interstitial space of a given cluster of microspheres (mesopores, 2–50 nm in diameter), or even within the microspheres themselves (micropores, b2 nm in diameter). Typical values for surface area of the imprinted polymers are in the range of 100 to 400 m²/g. For pore size distribution there are both macropores and mesopores in the range 2 to 100 nm, and micropores of 0.6 to 2 nm in diameter. The most effective variables that control surface area and pore distributions are the percentage of crosslinking monomer, the type and amount of porogen, and the reaction temperature. Although binding and selectivity by MIPs in chromatographic or batch rebinding mode are not dependent on macroporosity, applications in drug delivery may rely on mass-transfer kinetics related to porosity. Surface area measurements in MIPs are primarily carried out using a nitrogen adsorption porosimeter using a BET (Brunauer, Emmett and Teller) analysis routine that is standard to all instruments. For pore size distributions in MIPs, the same nitrogen adsorption data can be analyzed using BJH (Barret, Joyner and Halenda) methods also available on porosimetry instruments. Results from this type of characterization are provided in table 1 which compares the effect of different porogens on surface area, pore volume, and average pore size in MIPs made with EGDMA/MAA monomers using lphe- an as template^[69].

Fig. 11: Model of morphology formation that provides the porous network in MIPs

Table 1. surface area pose volume and average pose size in MIPs made with EGDMA/MAa monomers using L-phen as template

Characterization of MIPs by spectroscopic analysis techniques

FT-IR and solid state NMR methods are useful for the measurement of functional group incorporation, especially for the quantification of the degree of polymerization and reactivity for each type of polymerizable group on the monomers. For example, quantitative FT-IR can be used to measure the extent of unreacted double bonds using the C–H out of plane bend at 900–950 cm^-1 and the –C-C– stretch at 1639 cm_1 ^[70]. A measure for the degree of polymerization is assessed from the number of unreacted double bonds, which are quantified by integration of the area under the peak corresponding to the wavelengths listed above. The integrated value is converted to number of double bonds using a calibration curve separately developed that correlates double bonds versus integration areas. Greater accuracy can be obtained if the ratio of peak areas for double bonds versus the area for the carbonyl group is used, eliminating any dependence on quantity of polymer used. Quantification of other functional groups can be done in similar fashion to measure the incorporation of different functional monomers. A more quantitative measure of overall unreacted double bonds in the different MIP materials can be obtained directly by 13C CP-MAS NMR (fig 11), without the need for calibration curves. All other functional groups of interest that are carbon-based, can also be quantified using this technique. As can be seen in fig 11, resonances around 100–130 ppm are due to double bonds of unreacted EGDMA; and peaks around 160–180 ppm are attributable to the different CjO groups that can be found in EGDMA and MAA ^[71]. The percentage of unreacted double bonds are calculated using the ratio of the integrated intensity of the carbonyl resonance assigned to the unsaturated carbonyl, relative to the total integrated intensity of the saturated carbonyl: e.g. 167 versus 175 ppm for EGDMA. According to literature precedent ^[72] the relaxation times for C=O and C=C bonds used in this study should be directly comparable due to similar cross-polarization behavior ^[73]. Furthermore, studies have determined optimum cross-polarization times to be in the range of 1–5 ms, which is necessary to maximize signal intensity and small deviations from optimum contact time will not effect quantitative measurements of peak areas ^[74].

Fig 12. Example of 13c CP-MAs NMR Spectra for imprinted polymers formulated a X/M ratio of 4/1, EGDMA/MAA

Characterization of MIP swelling

Swelling in MIPs has most often been measured using volumetric methods published by Sellergren and Shea^[69] . There are some difficulties, however, due to buoyancy (i.e. the polymers float) especially for the polymers in chlorinated solvents; and general accuracy of volumetric methods. A more accurate technique that can be used measures changes in volume for a single bead ^[75] In this case, the size of individual polymer particle can be observed under a microscope in the absence and presence of solvent. The particles are then photographed in swollen and unswollen states, and the ratios in surface area calculated to give the percent swelling. In many cases the particles have irregular shape or else there are wide ranges of different sizes between the particles; therefore, it is best to follow the same particle from the swollen state to the dry state.

Application of mip

Molecularly templated materials in chemical sensing

Molecular imprinting is an exciting and promising technique that is being increasingly adopted as a platform for creating responsive materials like chemical sensors. Molecularly imprinted materials appear to offer an inexpensive, robust, and reusable alternative to expensive and labile biorecognition elements. These materials can also exhibit binding affinities that are comparable to antibody/antigens, yet they offer the ability to design recognition sites for a plethora of analytes, including those for which biorecognition elements do not exist. Specificity for molecularly imprinted materials for their target analyte in comparison to structurally similar molecules is good, but they do not yet beat biorecognition elements. Molecularly imprinted materials offer an opportunity to design suites of materials/recognition elements for the same target analyte with unique binding characteristics and subsequent responses. Recently, several research groups have successfully coupled the low detection limits offered by fluorescence-based measurement with molecularly imprinted materials to develop totally self contained sensor elements. Thus, researchers have developed systems based on molecularly templated materials wherein there are no secondary reagents required and a selective and reversible analyte-dependent response is shown. The full potential of molecularly templated materials in chemical sensing awaits their extension to the detection of a wider variety of analytes and their use in analysis of real samples, fundamental information on the relationship between precursor chemistry and templating variables on the final material properties, and the chemistry that actually occurs within a molecularly imprinted material upon analyte binding/dissociation.

Molecularly imprinted polymers for robust food analysis

Nowadays society is highly sensitive towards contaminants in foodstuff. Such substances may be pesticides, herbicides, hormones and antibiotics. For the detection of contaminants fast analytical tools have to be developed. MIPs – providing selectivity and durability – can be employed for either extraction of the eligible group of analytes or for the actual separation of the imprinted species from the rest of the components. It has been demonstrated that MIPs are applicable for the determination of food additives, such as carbohydrates, peptides or flavor additives. Galactose, fructose and mannose as well as glucose have been used as templates, or amino acids and proteins like ribonuclease A or transferrin, and also caffeine. Furthermore vitamins and nucleotides can specifically be analyzed utilizing MIPs. A semi-covalent approach of molecular imprinting has been applied to cholesterol, where the imprinting took place covalently and the later recognition non-covalently. Another main topic was the imprinting of pesticides and herbicides and their detection in food and water with MIPs. Therefore MIPs have been applied for the determination of e.g., atrazine with sensors and assays or for investigating 2,4 dichlorophenoxyacetic acid contamination ^[76,77,78]. Furthermore, drugs   like local anaesthetics ^[79] and in particular anti-  biotics have been employed as template molecules,  such as macrolide antibiotics, chloramphenicol  ^[80,81] or clenbuterol ^[82]. Even surface imprints of  bacterial cells have been realized. ^[83]

Molecularly imprinted polymers in separation science

From an analytical separation point of view, a MIP may be best characterised as being a material, which in addition to the imprinted affinity sites contains both polar and lipophilic surface functionality. Thus, retention in chromatographic and membrane separation systems is due to a mixed-mode mechanism involving both selective affinity binding with imprints and non-specific physicochemical adsorption on polymer surface. Accordingly, the optimization of a MIP-based separation requires a fundamental understanding of the strength and nature of imprint–analyte and polymer surface–analyte interactions, respectively, and how these vary with the type of solvent or buffer employed. Unfortunately, imprint polyclonality prevents us from using simple one-site models for calculation of a single affinity constant and instead more sophisticated models for characterisation of imprint affinity distribution are required. Consequently, while chromatography provides an excellent means for the initial characterization of MIP selectivity, a more in-depth characterization of binding affinity distribution requires equilibrium binding experiments over a large range of ligand concentrations

Imprinted hydrogels as carriers for controlled release

Recently the attempts have been made to use environmentally responsive, MIP hydrogels as carriers for controlled release of therapeutic compounds. Hydrogels have been used as prime carriers for pharmaceutical applications, predominantly as carriers for delivery of drugs, peptides or proteins. They have been used to regulate drug release in reservoir-based, controlled release systems or as carriers in swellable and swelling-controlled release devices ^[84,85,86]. Hydrogels can be rendered sensitive to physiological conditions due to the presence of specific functional groups along their backbone polymer chains. The swelling behavior and associated release kinetics of these gels may be dependent on pH, temperature, ionic strength, or even drug concentration ^[86]. The technology has evolved to achieve sustained delivery of large molecules such as high molecular weight peptides for long periods of time (e.g. days and months), and certain groups have recently used MIP polymers to give sustained/controlled release of small-molecular weight drugs for therapeutic purposes. For example, PEG hydrogels as contact lenses have been composed with imprinted sites for the drug timolol ^[87]. The interaction of the imprinted sites with timolol must be overcome by the uptake of water and this slows the release of this drug into the eye. Thus, MIP contact lenses exhibited longer release times than non-imprinted polymers loaded with the drug ^[87]. The overall application of molecularly imprinted polymers are shown in the following table

Analyate class	Target analysis	Templete	Application	Polymer Material(s)
Pharmaceuticals	Penicillin G	Penicillin G Potassium salt	Binding Assay	Methcrllic acid 2-hydroxyethyl metnacrylatemethcrylamide
	Penicillin G	Penicillin G procaine salt	Solid phase extraction	1-(4-vinylphenyl)-3-[3,5-bis(tri fluromethyl)phenyl] urea methacrylamide
	Methadone	Methadone	Chromatography	Methacrylic acid, hydroxyethyl methacrylate Itaconic acid
Vitamins	Folic acid	N-Z-L-glutamic acid	Binding	2-amino-6-methylpyridine9-(3/4-vinylbenzene)adenine methacrylamide 2-methacrylamidepyridine 1-(4-vinylphenyl)-3-(3-nitrophenyl)urea
Amino acid	L-Hystidine	L-Hystidine	Sensor	Phenyltrimthoxysiline Methyltrimethoxysiline
	L-Proline	L-Proline	Sensor	Methacrylic acid
Nucleotide base	Cytosine,Thymine,guanine, adenine	9-Ethyladenine	Chromatography	Methacrylic acid
Pesticides	Monocrotophos	Monocrotophos	Solid-phase extraction	Acrylimide Methacrylic acid Acrylic acid
Proteins	Lysozyme	Lysozyme	Binding assay	Acryluic acid

Table 2 Design and Application example of molecularly imprinted polymer

Future Prospects

Current Limitations Of Mip And Ideal Drug Delivery Systems

The field of imprinted polymer drug delivery systems is still relatively new but is attracting increasing attention. Imprinted polymers that are sensitive to highly specific chemical stimuli are perhaps the materials under most rapid development, as these have the potential to act as truly smart medical systems, but many practical issues still remain. These are primarily a result of the difficulties in controlling MIP synthesis, with concomitant uncertainties relating to polymer architecture, chemical environment diversity and recognition site specificity. The optimisation, using predictive tools, of the nature and amount of functional monomers should overcome the time and material consuming method of “trial and error” and improve the specificity and the affinity of the template ^[88]. This may be achieved with the use of rapid experimental screening processes ^[89], combinatorial libraries ^[90] and with the application of isothermal titration calorimetry analysis, which has been proven to be a suitable method to investigate the thermodynamics of molecular recognition and to assess the efficiency of the molecular imprinting process ^[91].

 Recent developments in controlled polymer synthesis, especially in living free radical polymerisation chemistries ^[92], are now allowing much greater control over macromolecular architectures in functional materials. Other controlled polymerisation methods such as ring-opening metathesis polymerisation (ROMP) ^[93,94] have also been applied to MIP synthesis ^[95,96], while ROMP catalysts are now tolerant to the wide range of functional groups that are commonly used in imprinting chemistry ^[97,98]. However, there is still not one single, universally applicable technique for the controlled assembly of polymer matrices in three dimensions. Factors such as the gel effect during crosslinking contribute to the heterogeneity of imprinted matrices and the restricted mobilities and high local viscosities towards the latter stages of MIP synthesis can lead to residual reactive groups in the final material. In order to effect fine control over drug release kinetics, much tighter definition of binding sites than can currently be achieved is necessary, combined with a reduction in matrix heterogeneity and tortuosity. It is also desirable to control pore size and volume in these cross-linked gels, perhaps in a way that allows the pores to be opened and closed in response to either a local change in conditions or an external signal. One way to reduce matrix heterogeneities is to prepare imprinted polymers as self-assembled monolayers or ultra-thin films on a well-defined support matrix. The recent work of the group of Sellergren in generating imprinted surfaces via the attachment of azo-initiators, iniferters and polymerisable groups to surfaces ^[99] is an important step toward well-defined imprinted films ^[100,101]. Although the driving force behind the development of these films was not drug delivery but the desire to recognise macromolecules and biopolymers, nevertheless imprinted films could find much use in drug delivery applications. This especially applies to the ever-expanding field of biopolymer drugs since diffusion of macromolecules is inherently disfavored from highly cross-linked matrices. Secondly, imprinted polymer films could in theory is prepared on existing formulated tablets or using existing drug delivery matrix materials. Since the vast majority of successful drugs are currently administered via the oral route (over 75% of the top selling drugs in the USA fit into this category) it is particularly desirable that an imprinted polymer drug delivery system can be adapted into a tablet-based dosage form. For the oral route there are further constraints on an imprinted polymer in that the delivery system must protect the therapeutic through the GI tract – an extremely challenging biochemical environment – in addition to being able to release the drug in the correct dose and only at the target site. For biopolymer drugs such as peptides and proteins this need for protection during in vivo transport is particularly important. An imprinted polymer able to accomplish all these tasks effectively is still a long way from being designed, let alone synthesised^.[101,102]

Conclusion:

The synthesis of molecularly imprinted polymers is a chemically complex pursuit and demands a good understanding of chemical equilibria, molecular recognition theory, thermodynamics and polymer chemistry in order to ensure a high level of success. Furthermore, optimization of the imprinted products is made more difficult by the fact that there are many variables to consider, some or all of which can potentially impact upon the chemical, morphological and molecular recognition properties of the imprinted materials. Initial speculation on MIP binding applications was that they would serve as synthetic antibodies; that is, they would provide specific binding for molecular targets in the same way antibodies do. MIPs have large numbers of different sites with a distribution of affinities that gives different average binding affinities depending on the number of sites accessed. Fortunately, in many instances it is possible to rationally predict how changing any one such variable, e.g. the cross-link ratio, is likely to impact upon these properties. Furthermore, a clear understanding of the underlying principles of simple free radical polymerization processes, especially when applied to macroscopic network polymers, provides a good basis to make such predictions. Macroporous polymers, which have permanent pore structures even in the dry state, are particularly attractive matrices for imprinting not least because of their appealing mechanical properties; however, binding can still only be indirectly modeled at the molecular level using the Evaluation methods. Therefore, applications of antibodies which are molecular in nature, may not be directly replaceable by MIPs which are amorphous bulk materials in nature. It may be more fruitful instead to adjust applications or formats to exploit MIP capabilities. Finally, the chemical, morphological and molecular recognition properties of molecularly imprinted polymers can be conveniently characterised by a complementary array of increasingly powerful analytical techniques, Drug delivery may be one of the best new applications of MIPs in this regard.

Reference:

[1]  M.V. Polyakov, Adsorption properties and structure of silica gel Zhur, Fiz. Khim. 2 (1931) 799–805.

[2] F.H. Dickey, Specific adsorbents, Proc. Natl. Acad. Sci. 35(1949) 227– 229.

[3] G. Wulff, A. Sarhan, U¨ ber die Anwendung von enzymanalog gebauten Polymeren zur Racemattrennung, Angew. Chem. 84 (1972) 364

[4] R. Arshady, M. Mosbach, Synthesis of substrate-selective polymers by host–guest polymerization, Macromol. Chem. Phys.-Makromol. Chem. 182 (1981) 687–692.

[5] M.J. Whitcombe, M.E. Rodriguez, P. Villar, E.N. Vulfson, A new method for the introduction of recognition site functionality into polymers prepared by molecular imprinting—synthesis and characterization of polymeric receptors for cholesterol, J. Am. Chem. Soc. 117 (1995) 7105– 7111

[6] G. Wulff, T. Gross, R. Scho¨ nfeld, Enzyme models based on molecularly imprinted polymers with strong esterase activity, Angew. Chem., Int. Ed. Engl. 36 (1997) 1962– 9164.

[7] C. Lu¨bke, M. Lu¨bke, M.J. Whitcombe, E.N. Vulfson, Imprinted polymers prepared with stoichiometric template– monomer complexes: efficient binding of ampicillin from aqueous solutions, Macromolecules 33 (2000) 5098–5105.

[8] P. Manesiotis, A.J. Hall, M. Emgenbroich, M. Quaglia, E. de Lorenzi, B. Sellergren, An enantioselective imprinted receptor for Z-glutamate exhibiting a binding induced colour change, Chem. Commun. (2004) 2278– 2279.

[9] B. Sellergren, Imprinted dispersion polymers. New easily accessible affinity stationary phases, J. Chromatogr. 673 (1994) 133.

[10] F.L. Dickert, O. Hayden, Molecular imprinting in chemical sensing, Trends Anal. Chem. 18 (1999) 192–199.

[11] G. Wulff, J. Vietmeier, H.G. Poll, Enzyme-analog built polymers: 22. Influence of the nature of the cross-linking agent on the performance of imprinted polymers in racemicresolution, Makromol. Chem.–Macromol. Chem. Phys. 188 (1987) 731– 740.

[12] B. Sellergren, Polymer- and template-related factors influencing the efficiency in molecularly imprinted solid-phase extractions, Trends Anal. Chem. 18 (1999) 164– 174.

[13] S.R. Tonge, B.J. Tighe, Responsive hydrophobically associating polymers: a review of structure and properties, Adv. Drug Deliv. Rev. 53 (2001) 109– 122.

[14] C. Alvarez-Lorenzo, A. Concheiro, Molecularly imprinted polymers for drug delivery, J. Chromatogr., B 804 (2004) 231– 245.

[15] R. Langer, N.A. Peppas, Advances in biomaterials, drug delivery, and bionanotechnology, AIChE J. 49 (2003) 2990– 3006.

[16] D.X. Cui, H.J. Gao, Advances and prospects of bionanomaterials, Biotechnol. Prog. 19 (2003) 683– 692.

[17] F.W. Billmeyer, Textbook of Polymer Science, Wiley, New York, 1984;

 [18] J. Brandrup, E.H. Immergut, E.A. Grulke, in: Polymer Handbook, fourth ed., Wiley, New York, 1999.

[19] R.Z. Greenley, J. Macromol. Sci.Chem. A14 (1980) 445.

[20] W. Funke, Br. Polym. J. 21 (1989) 107.

[21] R.B. Merrifield, J. Am. Chem. Soc. 85 (1963) 2149.

[22] O. Ramström, L.I. Andersson, K. Mosbach, J. Org. Chem. 58 (1993) 7562

[23]. Takeda, K.; Kobayashi, T. Bisphenol A. imprinted polymer adsorbents with selective recognition and binding characteristics. Sci. Tech. Adv. Mater. 2005, 6, 165-171

[27] D.A. Spivak, J. Campbell, Systematic study of steric and spatial contributions to molecular recognition by non-covalent imprinted polymers, Analyst 126 (2001) 793– 797.

[28] G. Wulff, R. Kemmerer, J. Vietmeier, H.G. Poll, Chirality of vinyl polymers. The preparation of chiral cavities in synthetic polymers, Nouv. J. Chim. 6 (1982) 681–687.

[29] B. Sellergren, Molecular imprinting by noncovalent interactions. Enantioselectivity and binding capacity of polymers prepared under conditions favoring the formation of template complexes, Makromol. Chem. 190 (1989) 2703– 2711

[24] F. Lanza, B. Sellergren, Molecularly imprinted polymers via high-throughput and combinatorial techniques, Macromol. Rapid Commun. 25 (2004) 59–68.

[25] F. Lanza, B. Sellergren, Method for synthesis and screening of large groups of molecularly imprinted polymers, Anal. Chem. 71 (1999) 2092– 2096.

[26] T. Takeuchi, D. Fukuma, J. Matsui, Combinatorial molecular imprinting: an approach to synthetic polymer receptors, Anal. Chem. 71 (1999) 285– 290.

[30] M.P. Davies, V. De Biasi, D. Perrett, Approaches to the rational design of molecularly imprinted polymers, Anal. Chim. Acta 504 (2004) 7 – 14.

[31] B. Sellergren, M. Lepisto¨ , K. Mosbach, Highly enantioselective and substrate-selective polymers obtained by molecular imprinting utilizing noncovalent interactions. NMR and chromatographic studies on the nature of recognition, J. Am. Chem. Soc. 110 (1988) 5853–5860.

[32] M.J. Whitcombe, L. Martin, E.N. Vulfson, Predicting the selectivity of imprinted polymers, Chromatographia 47 (1998) 457– 464.

[33] J. Zhou, X.W. He, J. Zhao, H.M. Shi, A study of binding action and selectivity of trimethoprim molecular template polymer, Chem. J. Chin. Univ. 20 (1999) 204– 208.

[34] J. Zhou, X.W. He, Study of the nature of recognition in molecularly imprinted polymer selective for 2-aminopyridine, Anal. Chim. Acta 381 (1999) 85–91.

[35] I. Idziak, A. Benrebouh, F. Deschamps, Simple NMR experiments as a means to predict the performance of an anti-17hethynylestradiol  molecularly imprinted polymer, Anal. Chim. Acta 435 (2001) 137– 140.

[36] C. Luebke, M. Luebke, M.J. Whitcombe, E.N. Vulfson, Imprinted polymers prepared with stoichiometric template– monomer complexes: efficient binding of ampicillin from aqueoux solutions, Macromolecules 33 (2000) 5098– 5105.

[37] B. Castro, M.J. Whitcombe, E.V. Vulfson, R. Vasquez-Duhalt, E. Ba´rzana, Molecular imprinting for the selective adsorption of organosulphur compounds present in fuels, Anal. Chim. Acta 435 (2001) 83– 90.

[38] O. Slinchenko, A. Rachkov, H. Miyachi, M. Ogiso, N. Minoura, Imprinted polymer layer for recognizing double-stranded DNA, Biosens. Bioelectron. 20 (2004) 1091–1097.

[39] Y. Lu, H. Zhang, X. Liu, Study on the mechanism of chiral recognition with molecularly imprinted polymers, Anal. Chim. Acta 489 (2003) 33– 43.

[40] J.H.G. Steinke, I.R. Dunkin, D.C. Sherrington, A simple polymerisabe carboxylic acid receptor: 2-acrylamide pyridine, Trends Anal. Chem. 18 (1999) 159–164.

[41] H. Sanbe, K. Hoshina, J. Haginaka, K.-K. Kunimoto, Q. Fu, Chiral recognition based on (S)-nilvadipine-imprinted polymers and chiral recognition mechanism, Chromatography 23 (2002) 65–66.

[42] T. Ogawa, K. Hoshina, J. Haginaka, C. Honda, T. Tanimoto, T. Uchida, Screening of bitterness-suppressing agents for quinin: the use of molecularly imprinted polymers, Analyst 94 (2005) 353– 362.

[43] Q. Fu, H. Sanbe, C. Kagawa, K.-K. Kunimoto, J. Haginaka, Uniformly sized molecularly imprinted polymer for (S)-nilvadipine. Comparison of chiral recognition ability with HPLC chiral stationary phases based on a protein, Anal. Chem. 75 (2003) 191–198.

[44] K. Tanabe, T. Takeuchi, J. Matsui, K. Ikebukuro, K. Yano, I. Karube, Recognition of barbiturates in molecularly imprinted copolymers using multiple hydrogen bonding, J. Chem. Soc., Chem. Commun. (1995) 2303– 2304.

[45] A. Katz, M.E. Davis, Investigation into the mechanisms of molecular recognition with imprinted polymers, Macromolecules 32 (1999) 4113– 4121.

[46] X. Dong, H. Sun, X. Lue, H. Wang, S. Liu, N. Wang, Separation of ephedrine stereoisomers by molecularly imprinted polymers. Influence of synthetic conditions and mobile phase compositions on the chromatographic performance, Analyst 127 (2002) 1427– 1432.

[47] A.J. Hall, P. Manesiotis, J.T. Mossing, B. Sellergren, Molecularly imprinted polymers (MIPs) against uracils: functional monomer design monomer–template interactions in solution and MIP performance in chromatography, Mater. Res. Soc. Symp. Proc. 723 (2002) 11 – 15.

[48] F. Lanza, M. Ruther, A.J. Hall, C. Dauwe, B. Sellergren, Studies on the process of formation, nature and stability of .  binding sites in molecularly imprinted polymers, Mater. Res. Soc. Symp. Proc. 723 (2002) 93– 103.

[49] J.P. Rosengren, J.G. Karlsson, I.A. Nicholls, Enantioselective synthetic thalidomide receptors based upon DNA binding motifs, Org. Biomol. Chem. 2 (2004) 3374–3378.

[50] J.G. Karlsson, B. Karlsson, L.I. Andersson, I.A. Nicholls, The roles of template complexation and ligand binding conditions on recognition in bupivacaine molecularly imprinted polymers, Analyst 129 (2004) 456– 462.

[51] J. Svenson, N. Zheng, I.A. Nicholls, A molecularly imprinted polymer-based synthetic transaminase, J. Am. Chem. Soc. 126 (2004) 8554–8560.

[52] J. Svenson, J.G. Karlsson, I.A. Nicholls, 1H nuclear magnetic resonance study of the molecular imprinting of (_)-nicotine: template self-association, a molecular basis for cooperative ligand binding, J. Chromatogr., A 1024 (2004) 39– 44.

[53] T. Sagawa, K. Togo, C. Miyahara, H. Ihara, K. Ohkuboa, Rateenhancement of hydrolysis of long-chain amino acid ester by cross-linked polymers imprinted with a transition-state analogue: evaluation of imprinting effect in kinetic analysis, Anal. Chim. Acta 504 (2004) 37– 41.

[54] J. O’Mahony, A. Molinelli, K. Nolan, M.R. Smyth, B. Mizaikoff, Towards the rational development of molecularly imprinted polymers: 1H NMR studies on hydrophobicity and ion-pair interactions as driving forces for selectivity, Biosens. Bioelectron 20 (2005) 1884–1893.

[55] A. Katz, M.E. Davis, Investigations into the mechanisms of molecular recognition with imprinted polymers, Macromolecules 32 (1999) 4113– 4121.

[56] D.J. Duffy, K. Das, S.L. Hsu, J. Penelle, V.M. Rotello, H.D. Stidham, Binding efficiency and transport properties of molecularly imprinted polymer thin films, J. Am. Chem. Soc. 124 (2002) 8290–8296.

[57] B.J. Brune, J.A. Koehler, P.J. Smith, G.F. Payne, Correlation between adsorption and small molecule hydrogen bonding, Langmuir 15 (1999) 3987– 3992.

[58] H.S. Andersson, I.A. Nicholls, Spectroscopic evaluation of molecular imprinting polymerisation systems, Bioorg. Chem. 25 (1997) 203–211.

[59] J. Svenson, H.S. Andersson, S.A. Piletsky, I.A. Nicholls, Spectroscopic studies of the molecular imprinting self assembly process, J. Mol. Recognit. 11 (1998) 83– 86.

[60] S. Striegler, E. Tewes , Investigation of sugar-binding sites in ternary ligand–copper(II)–carbohydrate complexes, Eur. J. Inorg. Chem. (2002) 487–495.

[61] H. Guo, X. He, Study of the binding characteristics of molecular imprinted polymer selective for cefalexin in aqueous media, Fresenius’ J. Anal. Chem. 368 (2000) 461–465.

[62] L. Ping, R. Fei, X.L. Zhu, J.Z. Hu, C.W. Yuan, Studies on the preparation of l-2-chloromandelic acid-imprinted polymer and its selective binding characteristics, Acta Polym. Sin. 5 (2003)724– 727.

[63] S.A Piletsky, K. Karim, E.V. Piletska, C.J. Day, K.W. Freebairn, C. Legge, A.P.F. Turner, Recognition of ephedrine enantiomers by molecularly imprinted polymers designed using a computational approach, Analyst 126 (2001) 1826– 1830.

[64] S. Subrahmanyam, S.A. Piletsky, E.V. Piletska, B.N. Chen, K. Karim, A.P.F. Turner, dBite-and-switchT approach using computationally designed molecularly imprinted polymers for sensing of creatine, Biosens. Bioelectron. 16 (2001) 631– 637.

[65] I. Chianella, M. Lotierzo, S.A. Piletsky, I.E. Tothill, B.N. Chen, K. Karim, A.P.F. Turner, Rational design of a polymer for microcystin-LR using a computational approach, Anal. Chem. 74 (2002) 1288– 1293.

[66] N.W. Turner, E.V. Piletska, K. Karim, M. Whitcombe, M. Malecha, N. Magan, C. Baggiani, S.A. Piletsky, Effect of the solvent on recognition properties of molecularly imprinted polymer specific for ochratoxin A, Biosens. Bioelectron. 20 (2004) 1060– 1067.

[67]. Guyot, in: D.C. Sherrington, P. Hodge (Eds.), Synthesis and Separations Using Functional Polymers, John Wiley & Sons, New York,1989, pp. 1– 36.

[68] L. Lloyd, J. Chromatogr. 544 (1991) 201.

[69] B. Sellergren, K.J. Shea, Influence of polymer morphology on the ability of imprinted network polymers to resolve enantiomers, J. Chromatogr. 635 (1993) 31– 49.

 [70] K.J. Shea, D.Y. Sasaki, An analysis of small-molecule binding to functionalized synthetic polymers by 13CP/MAS NMR and FT-IR spectroscopy, J. Am. Chem. Soc. 113 (1991) 4109– 4120.

[71] M. Sibrian-Vazquez, Ph.D. Thesis, Design, synthesis, and applications of bio-derived crosslinking monomers for molecular imprinting, Louisiana State University and Agricultural College, Baton Rouge, Louisiana, 2003.

[72] R.G. Earnshaw, C.A. Price, J.H. O’Donnell, A.K. Whittaker, J. Appl. Polym. Sci. 32 (1986) 5337– 5344.

[73] T. Hjertberg, T. Hargitai, P. Reinholdsson, Macromolecules 23 (1990) 3080– 3087.

[74] J. Schaefer, E.O. Stejskal, R. Buchdahal, Macromolecules 10 (1977) 384–405.

[75] V.K. Sarin, S.B.H. Kent, R.B. Merrifield, Properties of swollen polymer networks. Solvation and swelling of peptide-containing resins in solid-phase peptide synthesis, J. Am. Chem. Soc. 102 (1980) 5463– 5470.

[76] K. Haupt, A.G. Mayes, K. Mosbach, Anal. Chem. 70 (1998) 3936.

 [77] E. Yilmaz, K. Mosbach, K. Haupt, Anal. Commun. 36(1999) 167.

[78] R.J. Ansell, K. Mosbach, Analyst 123 (1998) 1611.

[79] L. Schweitz, L.I. Andersson, S. Nilsson, J. Chromatogr. A (1997) 5396. 792 (1997) 401.

 [80] M. Siemann, L.I. Andersson, K. Mosbach, J. Antibiot. 50(1997) 88.

[81] R. Levi, S. McNiven, S.A. Piletsky, S.H. Cheong, K.Yano, I. (1999) 493. Karube, Anal. Chem. 69 (1997) 2017.

[82] V. Crescenzi, G. Masci, M. Fonsi, G. Casati, Proceedings of 8154. the American Chemical Society, Boston , MA ,1998.

[83] A. Aherne, C. Alexander, M.J. Payne, N. Perez, E.N. Vulfson, J. Am. Chem. Soc. 118 (1996) 8771.

[84] Peppas NA, Colombo P. Analysis of drug release behavior from swellable polymer carriers using the dimensionality index. J Controlled Release 1997;45:35–40.

[85] Peppas NA, Bures P, Leobandung W, Ichikawa H. Hydrogels in pharmaceutical formulations. Eur J Pharm Biopharm 2000;50:27–46.

[86] Peppas NA, Khare AR. Preparation, structure and diffusional behavior of hydrogels in controlled release. Adv Drug Deliver Rev 1993;11:1–35.

[87] Alvarez-Lorenzo CH, Hiratani H, Gomez-Amoza JL, Martinez-Pacheco R, Souto C, Concheiro A. Soft contact lenses capable of sustained delivery of timolol. J Pharm Sci 2002;91:2182–92.

[88] I.A. Nicholls, K. Adbo, H.S. Andersson, et al., Anal. Chim. Acta 435 (2001) 9.

[89] F. Lanza, B. Sellergren, Anal. Chem. 71 (1999) 2092.

[90] T. Takeuchi, D. Fukuma, J. Matsui,Anal. Chem. 71 (1999) 285.

[91] A.Weber, M. Dettling, H. Brunner,G.E.M. Tovar, Macromol. Rapid. Commun. 23 (2002) 824.

[92] K. Matyjaszewski, Controlling polymer structures by atom transfer radical polymerization and other controlled/living radical polymerizations, Macromol. Symp. 195 (2003) 25– 31.

[93] T.L. Choi, R.H. Grubbs, Controlled living ring-openingmetathesis polymerization by a fast-initiating ruthenium catalyst, Angew. Chem., Int. Ed. 42 (2003) 1743–1746.

[94] W.J. Feast, E. Khosravi, Synthesis of fluorinated polymers via ROMP: a review, J. Fluorine Chem. 100 (1999) 117–125.

[95] A. Patel, S. Fouace, J.H.G. Steinke, Novel stereoselective molecularly imprinted polymers via ring-opening metathesis polymerisation, Anal. Chim. Acta 504 (2004) 53– 62.

[96] A. Patel, S. Fouace, J.H.G. Steinke, Enantioselective molecularly imprinted polymers via ring-opening metathesis polymerisation, Chem. Commun. (2003) 88– 89.

[97] C.W. Bielawski, O.A. Scherman, R.H. Grubbs, Highly efficient syntheses of acetoxy- and hydroxy-terminated telechelic poly (Butadiene)s using ruthenium catalysts containing nheterocyclic ligands, Polymer 42 (2001) 4939– 4945.

[98] R.R. Schrock, Olefin metathesis by molybdenum imido alkylidene atalysts, Tetrahedron 55 (1999) 8141– 8153.

[99] C. Sulitzky, B. Ruckert, A.J. Hall, F. Lanza, K. Unger, B. Sellergren, Grafting of molecularly imprinted polymer films n silica supports containing surface-bound free radical nitiators, Macromolecules 35 (2002) 79– 91.

[100] B. Sellergren, B. Ruckert, A.J. Hall, Layer-by-layer grafting f molecularly imprinted polymers via iniferter modified upports, Adv. Mater. 14 (2002) 1204– 1208.

[101] B. Ruckert, A.J. Hall, B. Sellergren, Molecularly imprinted omposite materials via iniferter-modified supports, J. Mater. hem. 12 (2002) 2275–2280.

[102] R. Tomlinson, J. Heller, S. Brocchini, R. Duncan, Polyacetal– oxorubicin conjugates designed for pH-dependent degradation, ioconjug. Chem. 14 (2003) 1096– 1106.

About Authors:

Chetan R. Shelke, Pravin S. Kawtikwar, Dinesh M. Sakarkar, Nikhil P. Kulkarni

Chetan R. Shelke
M. Pharm. Scholar, Sudhakararao naik institute of pharmacy,pusad dist-yavatmal, maharashtra, india.
For Correspondence

Pravin S. Kawtikwar
HOD, Department of Industrial Pharmacy, Sudhakararao naik institute of pharmacy,pusad dist-yavatmal, maharashtra, india

Dinesh M. Sakarkar
Sudhakarrao Naik Institute of Pharmacy, Pusad. Dist: Yavatmal. 445 204. MS. India

Nikhil P. Kulkarni
Sudhakarrao Naik Institute of Pharmacy, Pusad. Dist: Yavatmal. 445 204. MS. India