Challenges for the effective molecular imprinting of proteins.

Molecular imprinting is a technique that is used to create artificial receptors by the formation of a polymer network around a template molecule. This technique has proven to be particularly effective for molecules with low molecular weight (<1500 Da), and during the past five years the number of research articles on the imprinting of larger (bio)templates is increasing considerably. However, expanding the methodology toward imprinted materials for selective recognition of proteins, DNA, viruses and bacteria appears to be extremely challenging. This paper presents a critical analysis of data presented by several authors and our own experiments, showing that the molecular imprinting of proteins still faces some fundamental challenges. The main topics of concern are proper monomer selection, washing method/template removal, quantification of the rebinding and reproducibility. Use of charged monomers can lead to strong electrostatic interactions between monomers and template but also to undesired high aspecific binding. Up till now, it has not been convincingly shown that electrostatic interactions lead to better imprinting results. The combination of a detergent (SDS) and AcOH, commonly used for template removal, can lead to experimental artifacts, and should ideally be avoided. In many cases template rebinding is unreliably quantified, results are not evaluated critically and lack statistical analysis. Therefore, it can be argued that presently, in numerous publications the scientific evidence of molecular imprinting of proteins is not convincing.

Anionic and cationic dextran hydrogels for post-loading and release of proteins.

In this study, post-loading of proteins in and release from chemically crosslinked dextran hydrogels exploiting reversible electrostatic interactions was investigated. Methacrylated dextran (Dex-MA) was co-polymerized with methacrylic acid (MA) or dimethylaminoethyl methacrylate (DMAEMA) to form negatively and positively charged hydrogels, respectively. Incubation of negatively charged hydrogels in a low ionic strength (10 mM HEPES, pH 7.4) solution of cytochrome C (isoelectric point (pI) 10.2) led to quantitative absorption of the protein in the hydrogel. BSA (pI 4.8) and myoglobin (pI 7.2) were post-loaded into positively charged gels at neutral pH and negatively charged gels at pH 5, respectively. Loading efficiency and protein distribution in the gels were dependent on network charge (maximum loading efficiency at 100-150 µmol charged monomer/g gel) and crosslink density (higher and more homogenous loading at lower crosslink density) and on the ionic strength during loading (lower but more homogenous loading at higher ionic strength). Diffusion controlled release of the loaded protein was triggered by incubation of the hydrogel in HEPES buffered saline (HBS) pH 7.4. The amount of released cytochrome C in HBS varied from 94% to 70% from gels containing 60 and 150 MA/g, respectively. Importantly, quantitative release was obtained in 1 M NaCl, indicating that post-loading led to neither the formation of insoluble protein aggregates nor irreversible immobilization of the protein in the matrix. ESI-MS analysis of the released cytochrome C revealed that post-loading did not result in oxidation of the protein, as opposed to loading during preparation of the gels. In conclusion, this paper shows that post-loading of proteins in dextran hydrogels and release exploiting reversible charge interactions can be applied for efficient loading of proteins that are negative, positive or neutral at physiological pH. Importantly, our data demonstrate that using this loading method no chemical modification to the protein occurred.

The effect of network charge on the immobilization and release of proteins from chemically crosslinked dextran hydrogels.

Size is the main protein characteristic that determines its release from non-degrading neutral hydrogels. The effect of network charge on the release of proteins has not been studied systematically so far. Therefore, we investigated the release of proteins from charged hydrogels that were obtained by co-polymerization of methacrylated dextran (Dex-MA) with either methacrylic acid (MA) or 2-N,N-dimethylaminoethyl methacrylate (DMAEMA). These hydrogels are stable under physiological conditions. The effect of incorporation of the charged monomers on hydrogel charge, equilibrium swelling, and release of model proteins was assessed at both low (10mM HEPES) and physiological ionic strength (HEPES buffered saline, HBS). Model proteins were chosen on the basis of their charge at physiological pH; bovine serum albumin (BSA, negatively charged), myoglobin (neutral), and cytochrome C (positively charged). Interestingly, as opposed to myoglobin, both charged proteins were fully immobilized in the networks with opposite charge by electrostatic interaction at low ionic strength. On the other hand, at physiological ionic strength, the percentage of immobilized protein depended on the charge density of the hydrogel. For all proteins, the diffusion coefficient of the mobile fractions was not affected by opposite network charge. However, the release rate of BSA from similarly (negatively) charged networks significantly increased when a relatively high amount of charged monomers was incorporated. We conclude that incorporation of charge in a hydrogel network is suited as a tool for the immobilization of proteins and triggered release by increasing ionic strength.

Molecularly imprinted polymer particles: synthetic receptors for future medicine.

Molecular imprinting is a relatively new and rapidly evolving technique used to create synthetic receptors; it also possesses great potential in a number of applications in the life sciences. Traditionally, molecularly imprinted polymers are prepared by bulk polymerization, followed by crushing and sieving to obtain polymer beads. However, several methods can be used to synthesize polymer micro- and nano-particles directly, thereby avoiding the time- and labor-consuming process of crush sieving. Possible applications are foreseen in enhanced drug loading, controlled drug delivery and drug targeting. This review describes the different methods of synthesis of molecularly imprinted micro- and nano-particles and discusses how these methods challenge the outstanding issues that molecular imprinting is facing today, thereby facilitating biomedical applications in the future.