Abstract
In this study, heparin-imprinted poly(hydroxyethyl methacrylate-N-[(3-dimethylamino)-propyl] methacrylamide) cryogel column (HpMIP) was synthesized for removal of Hp from human plasma using molecular imprinting technique. Hp removal studies were performed from both aqueous solution and human plasma. Selectivity studies were performed using fast protein liquid chromatography (FPLC) system. The obtained results showed that the HpMIP column can remove Hp from both aqueous solutions and human plasma samples selectively. Ninety per cent of Hp was removed from 28 U/mL of human plasma samples successfully. Non-imprinted cryogel column (NIP) and plane PHEMA column were also synthesized to compare selectivity and non-specific adsorption properties.
Introduction
Heparin (Hp) is a linear, unbranched, highly sulphated polysaccharide which is composed of disaccharide units consisting of an uronic acid 1,4 linked to a D-glucosamine unit. Molecular weight of Hp ranges from 5 to 40 kDa with an average molecular weight of about 15 kDa (CitationWarkentin 2003, Citation2004, CitationWarkentin and Greinacher 2004, CitationNoti and Seeberger 2005). It is only found in mastocytes and has served as an anticoagulant in heart disease for more than 60 years (CitationEngelberg 1984). Hence Hp has a rapid anticoagulant effect, it is used very common as a drug for thromboprophylaxis or treatment in many clinical situations, such as invasive procedures and cardiovascular surgery, dialysis, atrial fibrillation, acute coronary syndromes, peripheral occlusive disease, venous thromboembolism and during extracorporeal circulation (CitationChong 2003, CitationJang and Hursting 2005) Nevertheless, there may also be critical adverse effects such as Hp-induced thrombocytopenia (HIT), bleeding and osteoporosis affirmed very often as severe and potentially life-threatening condition (CitationWarkentin 2003, Citation2004, CitationWarkentin and Greinacher 2004). Accordingly, after exertion anticoagulant effect of Hp, removal of Hp from blood is very necessary. There are certain methods developed for the removal of Hp. Protamine, used as Hp antagonist for clinical use nearly at the same time with Hp, is administered very often as the method for removal. Moreover, it is also used for in vitro titration of Hp besides the neutralization of Hp anticoagulant action (CitationJacques 1973). Other polymeric structures used for removal of Hp and also protamine action amplifier called poly-L-lysine (CitationMa et al. 1992, CitationLa Spina et al. 2014). The last method to be mentioned for Hp removal is the use of immobilized heparinase for enzymatic degradation of Hp (CitationLanger et al. 1982). Unfortunately the methods used for removal of Hp from blood may cause many side effects, which can be dramatic and often can be lethal (CitationKaminsky et al. 2008).
In current study, Hp-imprinted super macroporous cryogel adsorbent system was prepared as an alternative method to remove Hp from human plasma. Although the different approaches have been reported for recognizing Hp using molecular imprinting technique (CitationYoshimi et al. 2013, CitationLi et al. 2013) there are no studies that use this technique for the selective removal of Hp from human plasma. Molecular imprinting technique is used for creating polymeric matrices with both shape and functional group memory to be used in molecular recognition. Molecularly imprinted polymer is constructed via interacting template molecule, to be imprinted, with the functional monomer on the surface of polymer or within polymerizable mixture. Molecular imprinting is a sub field of biomimetic, which is very popular recently (CitationWulff 1995). Currently, MIPs have been used effectively in a wide range of fields from separation sciences, purification, sensors to drug delivery and extracorporeal therapy in consequence of their unique nature (CitationHaginaka et al. 1999, CitationLozinsky et al. 2001, Citation2003, CitationWulff 2002, CitationPiletsky et al. 2006, CitationLi and Li 2007, CitationHaginaka 2008, CitationArrua and Alvarez Igarzabal 2011). Macroporous cryogels get great attention especially in biotechnology, biomedicine and pharmaceutics. Their large interconnected pores present great advantages in case of working with viscous media like blood, plasma and plant or animal tissue extract. Cryogels provide short diffusion path, low-pressure drop and very short residence time for both adsorption and elution (CitationDemiryas et al. 2007, CitationBereli et al. 2011). These properties of cryogels are very advantageous for purification of biologically relevant proteins (CitationDerazshamshir et al. 2010). In this study, Hp-imprinted poly(hydroxyethyl methacrylate-N-[(3-dimethylamino)-propyl] methacrylamide) cryogel column (HpMIP) was prepared, in which DMAPM was the cationic monomer and Hp was the template. The characterization studies were performed using swelling tests, FTIR, SEM, blood compatibility. Efficiency for selective removal of Hp from artificial solutions and human plasma was investigated. Selectivity of the adsorbent was shown by competitor molecules having similar chemical structures and FPLC studies were also performed.
Experimental
Materials
Hp (Cat No: 3393), chondroitin sulphate B (Cat No: 4384), human serum albumin (HSA), immunoglobulin G were obtained from Sigma (St. Louis, MO, USA), N-[3-dimethylamino)-propyl] methacrylamide (DMAPM), 2-hydroxyethyl methacrylate (HEMA) was purchased from Sigma, N,N,N,N-tetramethyl ethylene diamine (TEMED) and ammonium per sulphate (APS) were supplied by BioRad (Hercules, CA, USA), methylene-bis acrylamide (MBAAm) was from Acros (Geel, Belgium). Sodium phosphate dibasic and sodium phosphate monobasic were obtained from Merk AG (Darmstadt, Germany). All chemicals used in this study were analytical reagent grade. All water used in experiments was purified using a Barnstead (Dubuque, IA) ROpure LP® reverse osmosis unit. Buffer and sample solutions were prefiltered through a 0.2-μm membrane (Sartorius, Gottingen, Germany). All glassware were extensively washed with dilute nitric acid before use.
Synthesis of Hp-imprinted (HpMIP) cryogels
The cryogel columns were prepared partially frozen reaction systems, in which the water molecules formed ice crystals and played a role of pore maker. The gelation takes place in non-frozen part of this system. Once the gelation of non- frozen microsystem completed, ice crystals melted and formed big pores surrounded by thin gel walls (CitationLozinsky et al. 2003). HpMIP cryogel columns were synthesized as follows; 10.70 mmol of HEMA monomer and 1.80 mmol of MBAAm cross-linker were dissolved in DI water. The Hp–DMAPM complex was prepared by dissolving the Hp (template) and DMAPM (functional monomer) (1 mol Hp/500 mol DMAPM) in 1 mL of DI water. The complex mixture was hold in refrigerator (+ 4°C) for 2 h to complete precomplexation before use. Hp–DMAPM complex was added to HEMA/MBAAm monomer solution in an ice bath. The monomer mixture included 40 mg Hp a final monomer ratio of 10% (w/v). Then, free radical polymerization was initiated by TEMED (1%, w/w) and APS (1% w/w). This mixture was immediately poured into plastic syringes (3 mL, ID 1.5 cm) and frozen at –12°C for 16 h. The HpMIP cryogel column was left at room temperature in order to form interconnected macropores by defrosting ice crystal in cryogel structure. summarizes the optimization studies of HpMIP columns. For the optimization of cross-linker amount, the monomer solution was prepared using different mole-ratios of HEMA monomer and MBAAm cross-linker by the same procedure (). Once the best mole ratio between HEMA and MBAAm was determined, the other optimization studies were performed to get proper HpMIP cryogel. The optimization of mole ratios between the Hp and DMAPM monomer in complex mixture and the optimization of the complex amount in HpMIP column was investigated as well ().
Table I. The optimization of the HpMIP columns.
Each of synthesized HpMIP cryogel columns were washed with deionized water and ethanol/water solution (30/70, v/v) in order to remove unreacted monomers, and stored at 4°C until use in swollen state. The same procedure of HpMIP cryogel column was applied for preparation of NIP cryogels without using Hp molecule. NIP columns were used as control columns to evaluate the interactions between DMAPM molecules and Hp molecule having no specific cavities of Hp molecules. The plain PHEMA cryogel columns were also prepared for investigation of nonspecific interactions between Hp molecules and PHEMA cryogel as well. The plain PHEMA cryogel column was prepared same as the HpMIP cryogel column procedure without using any of Hp and DMAPM molecules.
Characterization of HpMIP and NIP columns
The swelling tests were performed as described in our previous work (CitationDerazshamshir et al. 2010). All measurements were performed three times and the average values were presented. The specific surface area of HpMIP and NIP cryogel columns in dry state was determined by multipoint Brunauer–Emmett–Teller (BET) apparatus (Quantachrome, Nova 2200E, USA). The morphology of HpMIP and NIP cross section was investigated by scanning electron microscope (SEM) (JEOL, JSM 5600, Tokyo, Japan). Fourier Transform Infrared (FTIR) spectra of Hp, DMAPM and Hp–DMAPM complex, and HpMIP and NIP cryogel columns were obtained using a FTIR spectrophotometer (FTIR 8000 Series, Shimadzu, Japan). Blood compatibility tests were studied in vitro to investigate the interactions between blood and HpMIP and NIP cryogel columns were investigated by measuring prothrombin time (PT) and active partial thromboplastin time (APTT) to evaluate the coagulation behaviour. HpMIP and NIP cryogel columns were equilibrated, using PBS buffer (0.1 M, pH 7.4) for 1 hand then washed with 0.5 M NaCl solution and DI water for several times at 37°C. A piece of HpMIP and NIP samples were incubated with 500 μL of freshly centrifuged human plasma for 30 min at 37°C. Then HpMIP and NIP samples were removed from plasma and PT and APTT values were measured using a semi-automatic blood coagulation analyzer (Tokra Medikal, Ankara, Turkey). Control samples were prepared using 50 μl of PBS. All measurements were performed for three times and the average of results were presented (CitationHe et al. 2010, CitationYıldırım et al. 2013).
Removal of template
The removal efficiency of template is essential to obtain desired amount of specific Hp cavities. For molecular imprinting technique the template-removal process should be planned properly for use of imprinted polymers in its full potential for desired application (CitationCheong et al. 2013). In order to remove the template (Hp) molecules from HpMIP cryogel columns, 100 mM acetate buffer at pH 4 was selected as eluent; 10 mL of elution solution was passed through column using a peristaltic pump (Watson-Marlow, Wilmington, MA, USA) for 2 h. Then DI water was passed through the cryogel column for half an hour. This procedure repeated until no Hp was observed in washing solution. The amount of Hp was determined using UV spectrophotometer at 280 nm. After this procedure, the template-free HpMIP cryogel columns were washed using DI water for several times.
Adsorption studies
Hp adsorption from aqueous solution
Adsorption studies were implemented in HpMIP column and in continuous system for 2 h at room temperature. The effect of initial Hp concentration was performed for each cryogel columns (HpMIPs, NIP, plain PHEMA) by changing Hp concentration in the range of 0.1 and 3.5 mg/mL. The effect of flow rate on adsorption capacity was studied by pumping 2.0 mg/mL of Hp solution in different flow rates (0.5–4.0 mL/min) for 2 h at room temperature.
All the adsorption experiments were performed three times and average of these data were used. The adsorption amount was calculated according to the following expression:
Here, Q is the adsorption amount of Hp in unit mass of HpMIP column (mg/g); Ci and Cf are the initial and the final Hp concentrations of the aqueous solutions in unit of mg/mL, respectively; V is the volume of aqueous solution in mL; m is the mass of dried HpMIP column in unit of g (CitationBaydemir et al. 2007). All experiments were performed using NIP column under same conditions as well.
Desorption and repeated use
Desorption of Hp molecules were studied by using the same solution chosen for template removal studies (100 mM Acetate buffer at pH 4); 20 mL of elution solution was pumped through the column for 2 h at room temperature. The Hp content of initial and final solutions determined by UV spectrophotometer at 280 nm. Desorption ratio was calculated according to the following equation:
In order to show the reusability of HpMIP cryogel column, adsorption–desorption cycles were repeated 10 times using the same column. These experiments were repeated for NIP column as well.
FPLC studies
Fast Protein Liquid Chromatography (FPLC) system was used for separation of Hp from aqueous solution and human plasma (AKTA-FPLC, Amersham Bioscience, Uppsala, Sweden). The measurements were performed at 280 nm. HpMIP and NIP cryogel columns were packed into GE Healthcare column (10/10, 19-5001-01) of FPLC system. The mobile phase A was 10 mM PBS buffer (pH 7.4) and mobile phase B was acetate buffer at pH 4 containing 1 M NaCl solution. The chromatographic separation was performed using a linear gradient method. All the FPLC studies were performed at 4.0 mL/min flow-rate. All buffers and protein solutions were filtered before use. For the gradient procedure, mobile phase A was passed through the column for 7 min. After starting period, a linear gradient was started applying mobile phase B from 0% to 100% in 1 min, continued for 10 min and equilibrated by mobile phase A 100% in last 3 min. The separation was performed at room temperature. Void volume was determined with KBr solution. The standard solutions of Hp, HSA, IgG and CHND were applied to HpMIP and NIP column, separately to maintain the retention times in various concentrations. Once observing the retention times of the substances, the human plasma samples were applied to the FPLC column. The plasma samples were obtained from a volunteer person. Plasma samples were overloaded with Hp and applied to the FPLC system to investigate the selectivity of the HpMIP column against Hp in natural environment. Plasma samples were prepared as described in (CitationHe et al. 2010). The samples were applied to the HpMIP column, with the dilution ratios of 1/2, 1/5, 1/10, 1/20 and 1/50.
Selectivity is the most important parameter to express the molecular recognition ability of the synthesized polymer (CitationBaydemir et al. 2007). The chromatographic performance of HpMIP and NIP cryogel column was investigated in the presence of the binary mixtures of Hp/HSA; Hp/IgG; Hp/CHND solutions. Plasma proteins; HSA and IgG were selected due to presence in human plasma. CHND was selected because its size and charge properties similar to Hp. Hp is a linear, unbranched and highly sulphated polysaccharide, which is known as one of the most acidic macromolecules in nature. At this point of view, the competitive compounds were selected according to their pI values and their acidic/basic characters at physiological pH 7.4. The molecular weight of these proteins was also taken into consideration to confirm the shape memory of imprinted cavities. At pH 7.4, HSA (pI 4.9) was negatively charged and the HIgG (pI: 6.4–9.0) has considerably negative charge at this pH. Initial concentrations of the binary mixtures were adjusted as; 1000 ppm Hp/300 ppm HSA; 1000 ppm Hp: 100 ppm IgG; 500 ppm Hp: 500 ppm CHND. Selectivity constants were calculated according to the retention times of the compounds, obtained using binary samples (CitationLaura et al. 2002).
Capacity factors (k’) for both HpMIP and NIP columns were calculated using following equation:
Here, tR is the retention time of compounds and t0 is the retention time of non-retained compounds corresponding to void volume of the column. The separation factor (α) is the ratio of capacity factor of Hp (k’Hp) to the capacity factor of competitive compounds (HSA, IgG and CHND) and can be expressed as
The imprinting factor (IF) was calculated as,
Here, k’MIP and k’NIP represent the capacity factor of the HpMIP and NIP columns, respectively. The selectivity factor (SF) was found using the ratio of IFs of the template molecule (Hp) to IFs of the competitive compounds and defined as
Theoretical plate numbers (N) is very important for characterization of HpMIP and NIP columns representing the column efficiency (CitationCormack and Mehamod 2013). N can be described by the following equation:
Here, tR is the retention time, W0.5 is the peak width at half height (in units of time). The measurement of backpressure of a chromatographic column at different flow rates is also crucial parameter for the characterization of physical property of column (CitationAlkan et al. 2009, Citation2010). It effects retention times of compounds especially in the case of working with viscous samples. Backpressure studies were also performed in FPLC system at different flow rates (2.0, 3.0, 4.5 mL/min).
Hp removal studies from human plasma
Hp removal studies were performed for HpMIP column using Hp-loaded human plasma sample. Hp, sodium salt, from porcine intestinal mucosa 140 USP/mg was obtained from Sigma (1U Hp is equal to 7.14 μg). In this part of the study, plasma samples were prepared in the concentration of 200 μg/mL (28 U/mL) and 71.4 μg/mL (10 U/mL) by adding Hp. Five millilitres of Hp-loaded plasma sample were circulated through HpMIP column for 2 h, at 1.0 mL/min, at room temperature. After adsorption procedure, the column was washed with 50 mM phosphate buffer at pH 7.4 for 5 min to remove non-specifically adsorbed molecules. The amount of Hp adsorbed onto HpMIP column was calculated using o-toluidine method (CitationZhu et al. 2005). The same procedures were performed for both NIP column and plain PHEMA column.
Results and discussion
Characterization studies
HpMIP and NIP cryogel columns were produced with 85% and 92% of gelation yields, respectively. They had similar swelling properties. For both HpMIP and NIP cryogels swelling ratios and the macroporosity values were obtained as 90% and 71%, respectively. The pore morphology in all columns has affected the flow rate of water through the column. If the cross linker amount is increased, monolithic cryogel columns gain more rigid and mechanically stable structure; therefore flow resistance of the columns was increased as well. HpMIP3 cryogel column was elected as a result of swelling studies due to the lack of mechanical stability. After macroporosity experiments, they lose their reshaping properties because of repeated squeezing. The surface areas of HpMIP8 and NIP columns were found as 48 and 42 m2/g, respectively. The surface area of the HpMIP and NIP columns are considerably higher than that of traditional cryogels produced by our group in previous studies (CitationAlkan et al. 2009, CitationKoç et al. 2011). According to SEM photos HpMIP and NIP columns were formed as thin polymeric walls and interconnected macropores (10–100 μm in diameter) (). This structure gave cryogel columns a unique property that interconnected pores constructed continuous channels through the columns so that mobile phase could flow inside easily. The polymeric wall seems as constructed by granules. This morphology may be due to the low solubility of MBAAm in polymerization media. The polymerization was conducted at − 12°C and the cross-linker solubility was decreased in these conditions. The granulated structure of cryogel leads to the higher surface area.
FTIR studies were performed for Hp, DMAPM and Hp–DMAPM complex to determine the structural evaluation of interaction between Hp and DMAPM molecules (). The OH stretching vibration can be seen between 3300 and 3350 cm− 1 for Hp and Hp–DMAPM complex. OH peak intensity of Hp was stronger than that of Hp–DMAPM. In DMAPM spectrum, the NH stretching vibration was observed at 3318 cm− 1. In Hp spectrum, the NH bending appears at 1635 cm− 1 and SO3 stretching peak appears at 1216 cm− 1, in the case of the complexation both the peaks were shifted through the left side at 1647 and 1225 cm− 1. This is an important point indicating that the Hp made complexation via these groups in its structure. shows FTIR spectrums of HpMIP (bottom) and NIP (upper) cryogels. For HpMIP the increase in peak intensity confirms the occurrence of template (Hp) in MIP cryogel. For instance; increase in the S = O symmetric stretch at 1154 cm− 1 (very strong), appearance asymmetric stretch at 1322 cm− 1 (medium), increase in the S–N stretch at 748 cm− 1, which corresponds to Hp. The intensity increased in O–H stretching in 3303 cm− 1 confirms the contribution of rich OH groups of Hp to MIP cryogel. Other peaks were commonly characteristic for cryogel as amide I at 1657 cm− 1, amide II at 1433 cm− 1, amide III at 1387 cm− 1, C = O stretching from PHEMA at 1717 cm− 1, C-H asymmetric and symmetric stretching at 2946 and 2878 cm− 1.
Biomaterials can be described as any materials interacting with living systems for use in medical devices or in particular applications for biological systems. All biomaterials have to meet the certain criteria before use for living systems. Depending on desired end use the blood compatibility, tissue compatibility, carcinogenicity, toxicity and mechanical stability tests must be performed using developed biomaterials (CitationDenizli and Pişkin 1995, CitationDenizli 1999, Citation2002, CitationErgün et al. 2012). If a material contact with blood, firstly the small molecules are floated on surface (i.e. water, ions); this event is followed by adsorption of plasma proteins onto surface. The amount and the type of protein affect the coagulation mechanisms deeply. That is why it is necessary to measure the PT, APTT for the determination of the blood compatibility of a biomaterial. The PT test shows the activation of the intrinsic coagulation factors; APTT test shows the activation of the extrinsic coagulation factors (). APTT times of HpMIP and NIP cryogel columns are apparently higher than that of control sample. HpMIP cryogel column was tested after template removal. For molecular imprinting technique the template removal studies is very important to get cavities in maximum amount. Unfortunately, template molecules cannot be removed completely due to the fact that they positioned inner parts of the polymeric materials. In this study, the template molecule is Hp and it is an anticoagulant; that's why HpMIP column shows higher APTT values than that of the others. The NIP cryogel columned show higher APTT time than that of control sample, due to the biocompatibility of HEMA.
Table II. Coagulation times of control, HpMIP and NIP cryogels.
Template removal studies
Template removal studies were performed to obtain the specific Hp cavities in MIP cryogel columns, which were used as free Hp rebinding sites. It is very important to find out proper solvent to remove template from the MIP material to have most effective rebinding sites (CitationKomiyama et al. 2003). Hp is a big and negatively charged molecule. To disturb the interactions between Hp and DMAPM molecules in HpMIP cryogel column 1M-NaCl-included acetate buffer (pH 4) was selected as solvent. The template was removed from the HpMIP2 (74.0%) cryogel column more efficiently than that do from HpMIP1 (63%) cryogel column. HpMIP1 column included higher amount of cross linker than that of HpMIP2 column (). Although the high amounts of cross linker do the imprinted polymers mechanically stable and insoluble and they also preserves the cavities, they can cause the binding sites very close to each other (CitationKomiyama et al. 2003). Once the polymeric structure gets more rigid and the 3D structure gets tight, the cavities may be placed inner parts and due to the high cross linker amount they cannot be released from the polymer easily, so HpMIP1 cryogel column released low amount of template than HpMIP2 cryogel column. The template and the functional monomer ratios in precomplexations were also varied to determine the template to functional monomer mole ratio (). The template removal ratios were obtained as 76%, 72% and 62% for HpMIP2, HpMIP4 and HpMIP5, respectively. The maximum template removal ratio was obtained for HpMIP2 with the 1:500, template (Hp) to functional monomer (DMAPM) mole ratio. According to results obtained, template removal ratios were decreased by increasing functional monomer ratio in the complex due to the stronger interactions. As expected, when mole ratio of functional monomers increased, interactions between the Hp and DMAPM getting stronger. Although the strong interactions are preferable to obtain proper cavities, it brings difficulties during the template removal step for molecular imprinting procedure.
Adsorption studies
Hp adsorption from aqueous solution
For molecular imprinting technique, the cavity amount in the synthesized polymers is one of the most vital parameters to achieve the maximum adsorption performance (CitationKomiyama et al. 2003). Effects of equilibrium concentration of Hp on amount of Hp adsorption onto HpMIP2, HpMIP6, HpMIP7 and HpMIP8 were investigated to find out the optimum amount of cavity for HpMIP columns (). The maximum Hp adsorption capacities were found as 2.44, 4.81, 9.207 and 9.36 mg/g for HpMIP2, HpMIP7, HpMIP8 and HpMIP9 cryogel columns, respectively (). According to these results, maximum Hp adsorption capacities were increased with increasing pre-complex amount in HpMIP2, HpMIP6, HpMIP7 and HpMIP8 columns. However, it is obvious that Hp adsorption capacity remained almost constant for HpMIP7 and HpMIP8, even with increasing pre-complex amount in HpMIP8. In the light of these data, it can be concluded that cavity amount per unit mass of polymer may increase Hp adsorption amount. But after a certain point, the cavity amount was increased very much and cavities can combine in polymeric structures and so cannot work effectively. Thus there was no significant change observed for Hp adsorption capacity. As a result of this study, HpMIP8 column was selected as the most efficient column for Hp removal studies. All studies were performed using this column and called HpMIP (instead of HpMIP8) for the following sections.
Effect of Hp concentration on the amount of Hp adsorption was given in . At the beginning, Hp adsorption amount of HpMIP cryogel columns were increased by increasing initial Hp concentration (0.1–1.5 mg/mL) (). At the saturation point (1.5 mg/mL Hp) all available cavities were occupied with Hp molecules and at that point all columns reached their maximum Hp adsorption capacities. After saturation point, adsorption capacity of HpMIP cryogel columns remained the same for higher initial Hp concentrations (2.0 and 3.5 mg/mL).
The effects of initial concentration of Hp on adsorption amount were also investigated for NIP and plain PHEMA column as well for evaluation of nonspecific interactions. According to , the maximum Hp adsorption capacities were found as 9.2 mg/g for HpMIP; 7.43 mg/g for NIP and 0.49 mg/g for PHEMA cryogel columns. NIP column includes the same amount of DMAPM functional monomer per unit polymer with HpMIP column without any preorganization and it acts as an affinity column to Hp molecules. In adsorption studies at pH 7.4, the positively charged tertiary amine groups in DMAPM molecules rapidly attract negatively charged Hp molecules. The maximum Hp adsorption capacity of NIP cryogel column is lower than that of HpMIP column. This can be explained by the preorganization. For the NIP cryogel column, the functional monomers were distributed randomly and might be the one Hp molecule would have blocked more DMAPM molecules during the adsorption process than that of HpMIP column. As a result of this hindrance effects, Hp molecules cannot be adsorbed in higher amounts onto NIP column. In case of HpMIP column, the 1–500 mol of Hp molecule to DMAPM monomer is very specific for one Hp molecule in Hp cavity in HpMIP cryogel column. Thus it can be concluded that preorganization of the HpMIP columns adsorbed Hp molecules in higher amounts than did NIP columns. As seen in adsorption amount of Hp in NIP cryogel column was considerably higher than plain PHEMA column due to the presence of DMAPM molecules in NIP columns. So, it can be concluded that the interaction between Hp molecules and plain PHEMA cryogel column is negligible.
The adsorption isotherms were used to characterize Hp adsorption behaviour of HpMIP column. Constants to be reached from the adsorption isotherms, give us valuable information about surface properties of adsorbents and the adsorption mechanism. In this study, Langmuir and Freundlich isotherms were chosen to describe the adsorption process from equilibrium adsorption data (CitationLabrou and Clonis 1995). The adsorption isotherms also provide comparison of experimental and theoretical adsorption behaviours. Langmuir adsorption isotherm proposes the monolayer adsorption of analyte onto adsorbent surface and describes the homogeneous adsorbent surfaces. According to this model, each of the analyte molecule was adsorbed onto the adsorbent with the same adsorption activation energy. Langmuir adsorption isotherm can be defined as,
where, Qe defines the adsorbed Hp amount at equilibrium onto HpMIP column, Ce is equilibrium Hp concentration in solution (mg/mL), b is the Langmuir adsorption constant (mg/mL), it defines the affinity of adsorbent to the analyte in the solution, Qmax (CitationFinette et al. 1997) is the theoretical maximum adsorption capacity of Langmuir adsorption isotherm (mg/g) which indicates the homogeneous monolayer coverage of surface by the adsorbed molecules. Freundlich adsorption isotherm model was used to describe for adsorption behaviour onto heterogeneous surfaces. It defines the non-ideal adsorptions. Freundlich adsorption isotherm can be defined by following equation:
According to Freundlich adsorption isotherm, adsorbed molecules have different adsorption activation energies (CitationKomiyama et al. 2003) and this energy can be evaluated by Freundlich adsorption capacity (Qf) value. The n constant in this isotherm describes the deviation from linearity of Hp adsorption (CitationUmpleby et al. 2001). The Freundlich and Langmuir constants and the experimental adsorption values were summarized in . According to these results, the adsorption behaviour of the HpMIP columns is well fitted with Langmuir adsorption isotherm rather than Freundlich. R2 value of the linear plotting of the Langmuir isotherm is higher (95%) than that of Freundlich adsorption isotherm (89%). The theoretical maximum adsorption capacity of Langmuir isotherm (11.8 mg/g) is closer to the experimental one (9.2 mg/g) where Freundlich isotherm (6.12 mg/g) is far away from experimental value.
Table III. Langmuir and Freundlich adsorption constants and correlation coefficients for Hp.
Desorption and repeated use
Desorption and the reusability studies have vital importance to prove stability of adsorbent produced and to control the cost of a process designed. Desorption step should be achieved in short time and analyte should be desorbed as much as possible. After tenth use of same HpMIP column, the maximum Hp adsorption capacity was decreased only 6.15%. Desorption efficiency was found over 98% for each cycle. This result demonstrates the stability of the HpMIP columns and it can be used for a process as affinity adsorbents several times.
FPLC studies
The FPLC studies were performed for selective separation of Hp from the aqueous binary Hp/competitive compound solutions. shows the separation of Hp through the HpMIP and NIP columns. As can be seen from these figures, Hp molecules were separated from binary solutions successfully. Retention time of Hp was observed at around 14.0 min., while the retention time of competitive compounds was observed at around 9 min. The imprinting constants were calculated regarding the retention times ( and ). The k’ values of Hp were found greater than competitive compounds (HSA, IgG, CHND) for both HpMIP and NIP columns. The k’ values calculated for NIP column indicate that all the compounds were interacted with NIP cryogel column as well. However, k’ values of Hp in HpMIP column were greater than that of NIP column, which indicates that the imprinted cavities are working excellent for Hp recognition in HpMIP column. The α values were calculated greater for HpMIP column than that for NIP cryogel column. IFs were calculated to compare specific and nonspecific interactions of Hp with HpMIP and NIP cryogel columns, respectively. IF values of Hp were found as equal or greater than unity, showing specific interactions between Hp and HpMIP column in case of existence of competitive molecules. SFs were calculated for evaluation of the selective Hp adsorption behaviour of HpMIP and NIP columns. According to the results obtained (all the results greater than unity), HpMIP cryogel columns were adsorbed Hp selectively as compared with NIP cryogel column.
Table IV. Selectivity coefficients of the HpMIP column.
Table V. Selectivity coefficients of the NIP columns.
Ns were also calculated for both HpMIP and NIP columns. Higher N values confirm the high chromatographic efficiency of columns. N values were found higher for Hp than competitors for both HPNIP and NIP cryogel column. However N values of Hp in HpMIP cryogel column were significantly higher than that of NIP cryogel column. So, the overall picture of the FPLC studies demonstrating clearly the efficiency of the Hp imprinting, means that HpMIP column can separate Hp from the other competitive compounds successfully. The Hp-removal studies were performed using Hp-loaded plasma samples via HpMIP connected FPLC systems. represents the Hp separation from human plasma. Initial plasma samples have 120 mg/mL initial Hp concentration. This value was selected as high concentration of Hp to observe separation peak of Hp clearly. Although it is very high concentration with respect to the clinical applications (CitationGaus and Hall 1998) for the 1/50 dilution ratio (240 μg/mL) we observed a good separation for Hp. It can be concluded that, Hp was separated from plasma samples successfully using HpMIP column. It should be noted that it is not possible to evaluate both unbound and bound peaks in chromatogram for lower concentrations of Hp in plasma samples (less than 200 μg/mL) (). Due to the characteristic properties of cryogels, backpressures of HpMIP column were observed very low. It was slightly increased as 0.1 ± 0.014; 0.175 ± 0.007; 0.25 ± 0.028; 0.36 ± 0.014 mPa by increasing flow rate from 2.0; 3.0; 4.0; 5.0 mL/min, respectively. The newly synthesized PHEMA-based Hp-imprinted cryogel column (NIP) was useful to study viscous samples at high flow rates and it promises a useful method for chromatographic separations.
Hp removal studies from human plasma
Hp removal performance of HpMIP column was investigated using Hp-infused human plasma samples (). Gauss et al. (1998) reported that in cardiovascular surgery the Hp doses was used as 2–8 U/mL commonly. In order to observe the Hp removal performance of HpMIP column for higher concentrations of Hp than that for clinical conditions the Hp was loaded to plasma samples in concentration of 200 μg/mL (28 U/mL) and 71.4 μg/mL (10 U/mL) of Hp-infused human blood plasma samples. As can be seen from , the Hp removal ratios in HpMIP cryogel column were found as 90% and 87% for 28 and 10 U/mL, respectively. These values showed that the Hp was removed from human plasma successfully using HpMIP cryogel columns. Hp removal ratio in NIP cryogel column was found as 85% and 80% for 28 and 10 U/mL, respectively. Although the Hp removal capacity of NIP column seems high, selectivity of NIP column was very poor as discussed in previous sections. Hp removal ratio was found negligible for plain PHEMA cryogel column for both plasma samples.
Table VI. Removal amounts of Hp from human plasma for HpMIP, NIP and plain PHEMA cryogel columns.
Conclusion
Macroporous cryogels get great attention especially in biotechnology, biomedicine and pharmaceutics due to the advantages of their large interconnected pores (short diffusion path, low-pressure drop etc.). Molecular imprinting technique is very attractive method for producing the polymers with specific recognition sites. In this study the newly synthesized HpMIP columns were created to remove Hp from human plasma selectively. Several methods have been reported for removal of Hp from human blood. CitationMa et al. (1992) proposed Poly(L-lysine) immobilized poly(ethylene-vinyl alcohol) (PEVAL)-coated polyethylene (PE) hollow fibres to remove Hp from human blood and reported maximum adsorption capacity as 16.9 ± 2.6 mg/m2 (3059 ± 471 units/m2); CitationLanger et al. (1982) designed a enzymatic system for extracorporeal therapy using heparinase enzyme immobilized blood filter and they achieved 100% removal of Hp by enzymatic degradation. Kaminski et al. (2008) reported pH-sensitive Genipin-Cross-Linked chitosan microspheres for Hp removal from aqueous solutions and they reported removal ratio as 100% for 5 mL of 200 μg/mL of Hp solution at pH 6.8 using 40 mg of microspheres. In another study La Spina and co-workers used L-Lysine functionalized PHEMA based cryogels and they achieved 87% (4330 IU) of Hp (CitationLa Spina et al. 2014). In current study Hp was removed selectively from human plasma using molecularly NIPs about 90% of the initial amount of Hp. In current study the results demonstrated that the proposed method might provide an alternative to studies reported in literature for selective removal of Hp from human plasma.
Declaration of interest
The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.
References
- Alkan H, Bereli H, Baysal Z, Denizli A. 2009. Antibody purification with protein A attached supermacroporous poly(hydroxyethyl methacrylate) cryogel. Biochem Eng J. 45:201–208.
- Alkan H, Bereli H, Baysal Z, Denizli A. 2010. Selective Removal of the autoantibodies from rhematoid artritis patient plasma using protein A carrying affinity cryogels.Biochem Eng J. 51:153–159.
- Arrua RD, Alvarez Igarzabal CI. 2011. Macroporous monolithic supports for affinity chromatography. J Sep Sci. 34:16–17.
- Baydemir G, Andaç M, Bereli N, Say R, Denizli A. 2007. Selective removal of Bilirubin with bilirubin-imprinted particles.Ind Eng Chem Res. 46:2843‐2852.
- Bereli N, Saylan Y, Uzun L, Say R, Denizli A. 2011. L-Histidine imprinted supermacroporous cryogels for cytochrome C recognition.Sep Purif Technol. 82:28–35.
- Cheong WJ, Yang SH, Ali F. 2013. Molecular imprinted polymers for separation science: a review of reviews. J Sep Sci. 36:609–628.
- Chong BH. 2003. Heparin-induced thrombocytopenia.J Thromb Haemost1:1471–1478.
- Cormack P, Mehamod FS. 2013. Molecularly imprinted polymer synthesis using raft polymerisation.Sains Malays. 42:529–535.
- Demiryas N, Tüzmen N, Galaev IY, Pişkin E, Denizli A. 2007. Poly(acrylamide-allyl glycidyl ether) cryogel as a novel stationary phase in dye-affinity chromatography. J Appl Polym Sci. 105: 1808–1816.
- Denizli A. 1999. Heparin-Immobilized Poly(2-Hydroxyethylmethacrylate based microspheres. J Appl Polym Sci. 74:655–662.
- Denizli A. 2002. Preparation of immune-affinity membranes for cholesterol removal from human plasma. J Chrom B. 772:357–367.
- Denizli A, Pişkin E. 1995. Heparin-immobilized polyhydroxyethylmethacrylate microbeads for cholesterol removal: a preliminary report. J Chromat B. 670:157–161.
- Derazshamshir A, Baydemir G, Andaç M, Say R, Galaev IY, Denizli A. 2010. Molecularly imprinted PHEMA based cryogels for depletion of hemoglobin. Macromol Chem Phys. 221:657–668.
- Engelberg H. 1984. Heparin and the atherosclerotic process. Pharmacol Rev. 36:91–110.
- Ergün B, Baydemir G, Andaç M, Yavuz H, Denizli A. 2012. Ion imprinted cryogels for in vitro removal of iron from beta-thalassemic human plasma. J Appl Polym Sci. 125:254–262.
- Finette GM, Mao QM, Hearn MT. 1997. Comparative studies on the isothermal characteristics of proteins adsorbed under batch equilibrium conditions to ion-exchange, immobilised metal ion affinity and dye affinity matrices with different ionic strength and temperature conditions. J Chromatogr A. 763:71–90.
- Gaus K, Hall EAH. 1998. Surface Plasmon Resonance sensor for heparin measurements in blood plasma. Biosens Bioelectron. 13: 1307–1315.
- Haginaka J, Sanbe H, Takehira H. 1999. Uniform-sized molecularly imprinted polymer for (s)-ibuprofen retention properties in aqueous mobile phase. J Chromatogr A. 857:117–125.
- Haginaka J. 2008. Monodispersed, molecularly imprinted polymers as affinity-based chromatography media. J Chromatogr B. 866:3–13.
- He Q, Zhang J, Chen F, Guo L, Zhu Z, Shi J. 2010. An anti- ROS/hepatic brosis drug delivery system based on salvianolic acid B loaded mesoporous silica nanoparticlesBiomaterials. 31:7785–7796.
- Jacques LB. 1973. Protamine: Antagonist to heparin. Can Med Assoc J. 108:1291–1297.
- Jang IK, Hursting MJ. 2005. When heparins promote thrombosis; review of heparin-induced thrombocytopenia. Circulation. 111:2671–2683.
- Kaminsky K, Zazakowny K, Szczubialka K, Nowakowska M. 2008. pH sensitive genipin-cross-linked chitosan microspheres for heparin removal. Biomacromolecules9:3127–3132.
- Koç I, Baydemir G, Bayram E, Yavuz H, Denizli A. 2011. Selective removal of 17β- estradiol with molecularly imprinted particle embedded cryogel system. J Hazard Mater. 192:1819–1826.
- Komiyama M, Takeuchi T, Mukawa T, Asanuma H. 2003. Molecular Imprinting: From Fundamentals to Applications. Weinheim: Wiley-VCH Verlag GmbH & Co. KGaA.
- La Spina R, Tripisciano C, Mecca T, Cunsolo F, Weber V, Matiasson B. 2014. Chemically modified poly(2-hydroxyethyl methacrylate) cryogel for the adsorption of heparin. J Biomed Mater Res Part B doi:10.2012/jbm.b.33104.
- Labrou NE, Clonis YD. 1995. The interaction of Candida boidinii formate dehydrogenase with a new family of chimeric biomimetic dye-ligands.Arch Biochem Biophys. 316:169–178.
- Langer R, Linhardt RJ, Hoffberg S, Larsen AK, Cooney CL, Tapper D, Klein M. 1982. An enzymatic system for removing of heparin in extracorporeal theraphy. Science217:261–263.
- Laura P, Prandi C, Coppa M, Sparnacci K, Laus M, Lagana A, et al. 2002. Uniformly sized molecularly imprinted polymers (MIPs) for 17β- estradiol. Macromol Chem Phys. 203:1532–1538.
- Li L, Liang Y, Liu Y. 2013. Designing of molecularly imprinted polymer-based potentiometric sensor for the determination of heparin. Anal Biochem. 434:242–246.
- Li W, Li S. 2007. Molecular imprinting: a versatile tool for separation, sensors and catalysis. Adv Polym Sci. 206:191–210.
- Lozinsky VI, Plieva FM, Galaev IY., Mattiasson B. 2001. The potential of polymeric cryogels in bioseparation. Bioseparation10:163–188.
- Lozinsky VI, Galaev IY, Plieva FM, Savina IN, Jungvid H, Mattiasson B. 2003. Polymeric cryogels as promising materials of biotechnological interest. Trends Biotechnol. 21:445–451.
- Ma X, Mohammad SF, Kim SW. 1992. Heparin removal from blood using poly(L-Lysine) immobilized hollow fiber. Biotechnol Bioeng40:530–536.
- Noti C, Seeberger PH. 2005.Chemical approaches to define the structure-activity relationship of heparin-like glycosaminoglycans.Chem Biol12:731–756.
- Piletsky SA, Turner NW, Laitenberger P. 2006. Molecularly imprinted polymers in clinical diagnostics-Future potential and existing problems. Med Eng Phys. 28:971–977.
- Umpleby RJ, Baxter SC, Chen Y, Shah RN, Shimizu KD. 2001. Characterization of MIPs using heterogeneous binding models.Anal Chem. 73:4584–4591.
- Warkentin TE, Greinacher A. 2004. Heparin-induced thrombocytopenia: recognition, treatment, and prevention: the Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy.Chest J. 126:311–337.
- Warkentin TE. 2003. Heparin-induced thrombocytopenia: pathogenesis and management. Br J Haematol. 121:535–555.
- Warkentin TE. 2004. Heparin-induced thrombocytopenia. Diagnosis and management. Circulation110:454–458.
- Wulff G. 1995. Molecular imprinting in cross-linked materials with the aid of molecular templates - A way towards artificial antibodies. Angew Chem 34:1812–1832.
- Wulff G. 2002. Enzyme-like catalysis by molecularly imprinted polymers. Chem Rev. 102:1–27.
- Yıldırım A, Özgür E, Bayındır M. 2013. Impact of mesoporous silica nanoparticle surface functionality on hemolytic activity, thrombogenicity and non-spesific protein adsorption. J Mater Chem. 1:1909–1920.
- Yoshimi Y, Sato K, Ohshima M, Pletska E. 2013. Application of the ‘gate effect’ of a molecularly imprinted polymer grafted on an electrode for the real-time sensing of heparin in blood. Analyst. 138:5121–5128.
- Zhu AP, Ming Z, Jian S. 2005. Blood compatibility of chitosan/ heparin complex surface modified ePTFE vascular graft. Appl Surf Sci. 241:485–492.