Glucose binding to molecularly imprinted polymers

The main goal of this study was to prepare molecularly imprinted polymers (MIPs) with glucose recognition sites and to evaluate their glucose-binding properties for potential applications in glucose sensing and self-regulating insulin delivery devices. To mimic glucose-binding sites of natural proteins, monomers possessing functional groups similar to amino acids were used. Vinyl acetic acid (VAA), acrylamide (AAm), 4-pentenoic acid (PA), and allyl benzene (AB) were copolymerized with a cross-linking agent (N,N'-methylenebisacrylamide, BIS) in the presence of glucose as a template. The binding affinity of glucose to MIPs was examined by using an equilibrium dialysis technique. The dissociation constants of the MIPs were determined by Scatchard analysis. MIPs showed glucose-binding affinity, while polymers synthesized in the absence of glucose template did not show a glucose-binding property. MIPs composed of VAA, AAm, PA, and AB at optimized mole ratios of monomers and cross-linker showed the highest glucose-binding affinity, KD = 1.66 mM, which is comparable to that of a well-known glucose binding protein, concanavalin A (KD = 1.84 mM). The affinity between monomer and glucose was in the order VAA > AAm > AB > PA.     

Glucose binding to molecularly imprinted polymers

The main goal of this study was to prepare molecularly imprinted polymers (MIPs) with glucose recognition sites and to evaluate their glucose-binding properties for potential applications in glucose sensing and self-regulating insulin delivery devices. Tomimic glucose-binding sites of natural proteins, monomers possessing functional groups similar to amino acids were used. Vinyl acetic acid (VAA), acrylamide (AAm), 4-pentenoic acid (PA), and allyl benzene (AB) were copolymerized with a cross-linking agent (N,N′-methylenebisacrylamide, BIS) in the presence of glucose as a template. The binding affinity of glucose to MIPs was examined by using an equilibrium dialysis technique. The dissociation constants of the MIPs were determined by Scatchard analysis. MIPs showed glucose-binding affinity, while polymers synthesized in the absence of glucose template did not show a glucose-binding property. MIPs composed of VAA, AAm, PA, and AB at optimized mole ratios of monomers and cross-linker showed the highest glucose-binding affinity, K _D = 1.66 mM, which is comparable to that of a well-known glucose binding protein, concanavalin A (K _D = 1.84 mM). The affinity between monomer and glucose was in the order VAA > AAm > AB > PA.     

生物可降解分子印迹聚合物的制备及评价

背景：交联剂是支撑分子印迹聚合物骨架的主要单元,分子印迹聚合物是否生物友好与交联剂的性能密不可分,但目前常用交联剂的生物相容性和生物降解性还不明确。 目的：制备新型生物可降解分子印迹聚合物,分析其吸附性能和可降解性能。 方法：以丙烯酰化的聚ε-己内酯为交联剂,以丙烯酸为功能单体,采用紫外光引发聚合法制备茶碱分子印迹聚合物,通过等温吸附、Scatchard分析和动力学曲线研究其吸附能力,在模拟人体生理环境体系中进行体外降解实验。 结果与结论：等温吸附曲线表明茶碱分子印迹聚合物和非分子印迹聚合物对模板分子茶碱均有吸附能力,但茶碱分子印迹聚合物的吸附量显著高于非分子印迹聚合物。茶碱分子印迹聚合物对茶碱的载药量为1.54%,包封率为12.48%,茶碱分子印迹聚合物和聚ε-己内酯二醇在观察时间内的体外降解率分别为6.60%和1.33%。制备的分子印迹聚合物不仅对目标分子有特异的吸附性能,而且具有良好的生物降解性能,可在模拟人体环境中进行降解。     

Biodegradable poly(polyol sebacate) polymers

We have developed a family of synthetic biodegradable polymers that are composed of structural units endogenous to the human metabolism, designated poly(polyol sebacate) (PPS) polymers. Material properties of PPS polymers can be tuned by altering the polyol monomer and reacting stiochiometric ratio of sebacic acid. These thermoset networks exhibited tensile Young's moduli ranging from 0.37+/-0.08 to 378+/-33 MPa with maximum elongations at break from 10.90+/-1.37% to 205.16+/-55.76%, and glass transition temperatures ranging from approximately 7-46 degrees C. In vitro degradation under physiological conditions was slower than in vivo degradation rates observed for some PPS polymers. PPS polymers demonstrated similar in vitro and in vivo biocompatibility compared to poly(L-lactic-co-glycolic acid) (PLGA).     

Development and characterisation of molecularly imprinted polymers based on methacrylic acid for selective recognition of drugs

Specific molecularly imprinted polymers (MIPs) for the drug reserpine (RES) using methacrylic acid (MAA) as the functional monomer were developed and characterised for the first time in this study. Evaluation of the various polymers by binding assays indicated that the optimum ratio of functional monomer to template was 4:1. Furthermore, the imprinting effect of the MIPs was assessed by the chromatographic method, which demonstrated that the MIPs had better chromatographic behavior and selectivity than those of the corresponding NIPs. A combination of BET, NMR, UV spectroscopy, and MISPE analyses for investigation of the imprinting and recognition properties revealed that strong specific interactions between the functional monomer and RES in the prepolymerization solutions and the aqueous solutions were probably responsible for RES recognition. The preparation of RES MIPs and elucidation of imprinting and recognition mechanisms may serve as useful references for other drug MIPs.     

Cytotoxicity of thermosensitive polymers poly(N-isopropylacrylamide), poly(N-vinylcaprolactam) and amphiphilically modified poly(N-vinylcaprolactam)

Thermosensitive polymers poly(N-isopropylacrylamide) (PNIPAM), poly(N-vinylcaprolactam) (PVCL) and PVCL grafted with amphiphilic poly(ethylene oxide) (PEO) chains (PVCL-graft-C11EO42) were prepared and characterized and their putative cytotoxicity was evaluated. The cytotoxicity of these thermosensitive polymers and their monomers was investigated as a function of polymer concentration, incubation time and incubation temperature by using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) cytotoxicity tests in Caco-2 and Calu-3 cell cultures. Also, the influence of the chain end functionality on toxicity was examined. Viability (MTT) and cellular damage (LDH) of the cells were shown to be dependent on the surface properties of the polymers, hydrophilicity or hydrophobicity. Hydrophilic PVCL and PVCL-graft-C11EO42 were well tolerated at all polymer concentrations (0.1-10.0 mg/ml) after 3 h of incubation at room temperature and at physiological temperature (37 degrees C). The more hydrophobic PNIPAM induced more clear cellular cytotoxicity at 37 degrees C. The monomers N-isopropylacrylamide and vinylcaprolactam and PEO-macromonomer showed dramatically higher cytotoxicity values with respect to the corresponding polymers. Cell damage was directly dependent on concentration, temperature and incubation time.     

Use of molecularly imprinted polymers in the solid-phase extraction of clenbuterol from animal feeds and biological matrices

Clenbuterol molecularly imprinted polymers (MIPs) as chromatographic stationary phase for the solid-phase extraction (SPE) of the drug from biological samples have been prepared. Propylene columns filled with 500 mg of clenbuterol MIPs have been tested with respect to their loading capacity, memory effects, selectivity toward related drugs (mabuterol, clenproperol, clenisopenterol, ritodrine) and specificity toward interferences arising from heterogeneous matrices such as animal feeds, bovine urine and liver. Analytes were concentrated on Extrelut 20 columns and the residues resuspended in 70% acetonitrile. Application, washing and elution fractions were collected and analyzed by HPLC-diode array detection. Results indicate this MIP approach in SPE is extremely selective for clenbuterol, mabuterol, clenproperol and clenisopenterol (>95% found in the eluate), with a loading capacity of about 20 microg/100 mg of stationary phase. Ritodrine showed a recovery rate of 51%. The molecular recognition mechanism is so specific to allow clenbuterol detection and identification by conventional detectors at level of interest (ppb) also from complex matrices such as feeds, urine and liver.     

Protein recognition via molecularly imprinted agarose gel membrane

Agarose gel membranes (AGMs), which could selectively recognize bovine serum albumin (BSA) and bovine hemoglobin (Hb), were prepared by molecular imprinting technique under moderate preparation conditions. Four imprinting processes, including gelation without any treatment, second gel-melting, and two glutaraldehyde crosslinking processes of fumigation or direct addition of the crosslinking agent, were developed to investigate the protein-recognition behavior of the AGMs. Results showed that the preparation processes affected the adsorption capacity and selectivity of the imprinted AGMs. Both BSA- and Hb-imprinted AGMs exhibited higher adsorption abilities for the targeted proteins (3.77-5.72 times for BSA, 1.31-2.18 times for Hb) than the nonimprinted ones. And the selectivity of BSA-imprinted AGMs for BSA molecules (the selective factor K = 3.29-4.90) was higher than that of Hb-imprinted AGMs for Hb (K = 0.32-1.17). The optimal adsorption capacity of BSA- and Hb-imprinted AGMs was 25.90 and 117.45 mg/g, respectively, when the membrane was crosslinked by glutaraldehyde with a fumigation process; the optimal selectivity of BSA- and Hb-imprinted AGMs was 4.91 when the membrane was prepared by second gel-melting process, and 0.76 when the membrane was prepared without any treatment. These findings demonstrate that the molecularly imprinted AGMs are hopeful to be used in specific protein analysis.     

Biomimetic glucose recognition using molecularly imprinted polymer hydrogels

Non-covalent molecular imprinting of poly(allylamine hydrochloride) (PAA.HCl) with D-glucose 6-phosphate monobarium salt (GPS-Ba) produced molecularly imprinted polymer hydrogels (MIP) having an affinity to glucose over fructose. The hydrogels were formed by ionic association of the template molecule, GPS-Ba, to the polymer, prior to covalent crosslinking using epichlorohydrin (EPI). The template was removed by an aqueous base wash. Batch equilibration studies using different MIP hydrogels and non-molecularly imprinted polymers (NIPs) were performed in aqueous and buffered media to determine the binding capacities and isomeric selectivities with respect to the sugars, glucose and fructose. MIP glucose hydrogels exhibited binding capacities in excess of 0.6g of glucose per g of dry gel in a 100% DI H(2)O glucose solution, and in a 50-50% glucose-fructose solution mixture. Equilibrium binding capacities of fructose were lower than those observed with respect to glucose, indicating an isomeric preference for the binding of glucose over fructose. These hydrogels demonstrated a remarkable degree of biomimetic sugar recognition to specifically and selectively bind glucose in their swollen state in environments mimicking physiological conditions.     

Hydrophilic polymers derived from vitamin E

Vitamin E containing copolymers for biomedical applications were obtained by copolymerization reaction of vitamin E methacrylate (VEMA) with 2-hydroxyethyl methacrylate (HEMA), N,N-dimethyl acrylamide (DMA) or vinyl pyrrolidone (VP), in different experiments. High molecular weight copolymers prepared by free radical reactions initiated by azobisisobutironitrilo, AIBN, present a random distribution of vitamin E derivatives along the macromolecular chains, and the average composition depends on the initial composition of the reaction medium. The relative flexibility of the polymeric systems was analyzed measuring the glass transition temperature of copolymeric sequences and that of the pure alternating diad (Tg12) obtained by the application of the treatments proposed by Johnston and Barton to all the systems. Tg12 was higher than the average Tg of both homopolymers (Tg) for the VEMA-HEMA system, Tg12 was lower than Tg for the VEMA-DMA system and Tg12 was similar to Tg for the VEMA-VP system. VEMA-HEMA copolymers gave rise to hydrogels in water, acidic and alkaline media. VEMA-DMA copolymers gave rise to hydrogels in acidic medium and dissolved in water and alkaline medium. VEMA-VP copolymers were soluble in all media. The swelling of all the hydrogels fit a second-order kinetics. A VEMA-HEMA hydrogel was selected for in vivo experiments in order to study the influence of vitamin E on the regeneration process of Achilles tendon. The polymeric derivatives of vitamin E stimulated the regenerative process as a consequence of the antiaging effect in the local area of application.     

Particulate retrieval of hydrolytically degraded poly(lactide-co-glycolide) polymers

This article describes a technique for the retrieval of polymeric particulate debris following advanced hydrolytic in vitro degradation of a biodegradable polymer and presents the results of the subsequent particle analysis. Granular 80/20 poly(L-lactide-co-glycolide) (PLG) was degraded in distilled, deionized water in Pyrextrade mark test tubes at 80 degrees C for 6 weeks. Subsequently, a density gradient was created by layering isopropanol over the water, followed by a 48-h incubation. Two opaque layers formed in the PLG tubes, which were removed and filtered through 0.2-micrometer polycarbonate membrane filters. In addition, Fourier transform IR spectroscopy (FTIR) was performed to confirm the presence of polymer in the removed layers. The filters were gold sputter coated, and scanning electron microscopy (SEM) images were made. FTIR analysis confirmed that the removed material was PLG. SEM images of the extracts from the upper (lowest density) opaque layer showed a fine, powderlike substance and globular structures of 500-750 nm. The SEM images of the lower (highest density) opaque layer showed particles with a crystalline-like morphology ranging in size from 4 to 30 micrometer. Particulate PLG debris generated with the described technique can be useful for further studies of its biological role in complications associated with poly(alpha-hydroxy)ester implants. This study shows the presence of very persistent nano- and microparticles in the degradation pathway of PLG.     

Hydrophilic polymers derived from vitamin E

Vitamin E containing copolymers for biomedical applications was obtained by copolymerization reaction of vitamin E methacrylate (VEMA) with 2-hydroxyethyl methacrylate (HEMA), N,N-dimethyl acrylamide (DMA) or vinyl pyrrolidone (VP), in different experiments. High molecular weight copolymers prepared by free radical reactions initiated by azobisisobutironitrilo, AIBN, present a random distribution of vitamin E derivatives along the macromolecular chains, and the average composition depends on the initial composition of the reaction medium. The relative flexibility of the polymeric systems was analyzed measuring the glass transition temperature of copolymeric sequences and that of the pure alternating diad (Tg12) obtained by the application of the treatments proposed by Johnston and Barton to all the systems. Tg12 was higher than the average T of both homopolymers (Tg) for the VEMA-HEMA system, Tg12 was lower than Tg for the VEMA-DMA system and Tg12 was similar to Tg for the VEMA-VP system. VEMA-HEMA copolymers gave rise to hydrogels in water, acidic and alkaline media. VEMA-DMA copolymers gave rise to hydrogels in acidic medium and dissolved in water and alkaline medium. VEMA-VP copolymers were soluble in all media. The swelling of all the hydrogels fit a second order kinetics. A VEMA-HEMA hydrogel was selected for in vivo experiments in order to study the influence of vitamin E on the regeneration process of Achilles tendon. The polymeric derivatives of vitamin E stimulated the regenerative process as a consequence of the antiaging effect in the local area of application.     

Hydrophilic modification of polysulfone via incorporation of poly(oxy-1,4-butanediyl) segments

Polysulfone was modified by incorporation of hydrophilic poly(oxy-1,4-butanediyl) [synonyms: poly(tetramethylene ether glycol), PTMG, poly(tetrahydrofuran), poly(THF), poly(tetramethylene oxide)] units into the polysulfone chain resulting in the formation of segmented block copolymers. These block copolymers were prepared by a melt transesterification procedure. The influence of the molecular weights of both, polysulfone and PTMG segments on the solid phase structure, the thermal behavior, the wetting behavior as well as the surface composition of the products was studied. It was shown that the incorporation of hydrophilic PTMG segments leads to a significant hydrophilization of polysulfone that partially results in an enhanced swelling ability.     

Hydrophilic, semipermeable membranes fabricated with poly(ethylene oxide)-polysulfone block copolymer

Semipermeable membranes may be fabricated from mixtures of poly(ethylene oxide)/polysulfone block copolymer (PEO-b-PSF) and polysulfone. Membranes fabricated with PEO-b-PSF possess a hydrophilic surface. PEO-b-PSF segregates to the membrane surface during phase inversion fabrication of the membrane rendering the surface hydrophilic. Changes in surface hydrophilicity were demonstrated by a dramatic reduction in the dynamic contact angle in water. With regard to the similar microporous hollow fiber membranes, a PEO-b-PSF membrane had a dynamic water contact angle of 33 degrees +/- 2 compared to a 111 degrees +/- 3 for a polysulfone membrane. Studies on porcine platelet-rich plasma in vitro demonstrated that the hydrophilic PEO-b-PSF membrane was resistant to platelet adhesion compared to a polysulfone membrane. An order of magnitude fewer adherent platelets were observed on a PEO-b-PSF membrane compared to a polysulfone membrane. The hydrophilicity of PEO-b-PSF makes it a unique material for the fabrication of membranes for medical devices.     

Hydrophilic interpenetrating polymer networks of poly(dimethyl siloxane) (PDMS) as biomaterial for cochlear implants

Poly(dimethyl siloxane) (PDMS) was bulk-modified to develop a new intra-cochlear electrode that can closely hug the inner wall of scala tympani (ST). The hydrophilicity of bulk and surface of PDMS was changed using a sequential method for preparation of interpenetrating polymer networks (IPNs). A series of IPNs, based on PDMS and poly(acrylic acid) (PAAc), was synthesized and characterized by means of attenuated total reflectance Fourier transform infrared spectroscopy, water contact-angle measurement, dynamic mechanical thermal analysis and peel strength tests. The performances of actual-sized fabricated electrodes were assessed inside a transparent model of ST, which was filled with saline. The cell behavior of L929 fibroblasts on materials was studied in vitro.     

Hydrophilic interpenetrating polymer networks of poly(dimethyl siloxane) (PDMS) as biomaterial for cochlear implants

Poly(dimethyl siloxane) (PDMS) was bulk-modified to develop a new intra-cochlear electrode that can closely hug the inner wall of scala tympani (ST). The hydrophilicity of bulk and surface of PDMS was changed using a sequential method for preparation of interpenetrating polymer networks (IPNs). A series of IPNs, based on PDMS and poly(acrylic acid) (PAAc), was synthesized and characterized by means of attenuated total reflectance Fourier transform infrared spectroscopy, water contact-angle measurement, dynamic mechanical thermal analysis and peel strength tests. The performances of actual-sized fabricated electrodes were assessed inside a transparent model of ST, which was filled with saline. The cell behavior of L929 fibroblasts on materials was studied in vitro.     

Adsorptive separation of hemoglobin by molecularly imprinted chitosan beads

A simply hemoglobin (Hb) molecularly imprinted polymer (MIP) was prepared using Hb as the imprinted molecule, acrylamide as the functional monomer and cross-linked chitosan beads as the supporting matrix. The MIP was achieved by entrapment of the selective soft polyacrylamide gel in the pores of the cross-linked chitosan beads by letting acrylamide monomer and the protein diffuse into the pores of chitosan beads before starting the polymerization. The chitosan beads were freed from the surrounding polyacrylamide gel by washing. The Langmuir and Freundlich adsorption models were applied to describe the equilibrium isotherms. Langmuir analysis showed that an equal class of adsorption was formed in the MIP and the adsorption equilibrium constant and the maximum adsorption capacity were evaluated. The MIP has much higher adsorption capacity for Hb than the non-imprinted polymer with the same chemical composition, and the MIP also has a higher selectivity for the imprinted molecule. The MIP can be reused in an easy way and the reproduction coefficient was approximately 100% at low concentration.     

牛血红蛋白分子印迹聚硅氧烷的吸附和释放性能

目的 制备对牛血红蛋白(HB)具有选择性吸附及缓释能力的生物材料.方法 以HB作为模版分子,以硅烷为功能单体,海藻酸钙水凝胶膜为载体合成HB分子印迹聚硅氧烷(MIP),同时不加模板HB制备非印迹聚硅氧烷(NIP),并研究其对HB的吸附和释放性能.结果 MIP对HB的重吸附能力明显强于NIP,并能选择性识别混合蛋白质中的模版蛋白质分子,同时在磷酸盐缓冲液(PBS)和Tris-HCl缓冲液中MIP对HB均具有很好的缓释能力.结论 通过分子印迹技术可制备对目标蛋白质具有选择性吸附及缓释能力的生物材料.     

Ocular release of timolol from molecularly imprinted soft contact lenses

The aim of this study was to evaluate "in vivo" the usefulness of molecular imprinting technology to obtain therapeutic soft contact lenses capable of prolonging the permanence of timolol in the precorneal area, compared to conventional contact lenses and eyedrops. Soft contact lenses (diameter 14 mm, center thickness 0.08 mm) consisted of N,N-diethylacrylamide (DEAA; main component of the matrix), methacrylic acid (MAA; functional monomer) and ethylene glycol dimethacrylate (EGDMA; cross-linker) were prepared by the conventional methodology (non-imprinted) or by applying a molecular imprinting technique using timolol as the template (imprinted ones). After washing and reloading, timolol release studies carried out in rabbits showed that the soft contact lenses made by the molecular imprinting method (34 microg dose) provided measurable timolol concentrations in the tear fluid for 2.0- and 3.0-fold longer than the non-imprinted contact lenses (21 microg dose) and eyedrops (doses of 34 and 125 microg), respectively. Furthermore, the area under the timolol concentration-time curve (AUC) was 3.3- and 8.7-fold greater for imprinted contact lenses than non-imprinted contact lenses and eyedrops, respectively. The timolol concentration of the eyedrops did not affect the precorneal residence time of drug significantly. On the other hand, timolol loading capacity of the contact lenses was improved by the molecular imprinting method; the sustaining of the drug levels in the tear fluid being proportional to the loading capacity of the contact lenses. These results indicate that imprinted soft contact lenses are promising drug devices able to provide greater and more sustained drug concentrations in tear fluid with lower doses than conventional eyedrops.