Ind. Eng. Chem. Res. 2010, 49, 9811–9814
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Protein Imprinting by Means of Alginate-Based Polymer Microcapsules Edgar P. Herrero,†,‡ Eva M. Martín Del Valle,† and Nicholas A. Peppas*,‡ Department of Chemical Engineering, UniVersity of Salamanca, Salamanca 37008, Spain, and Biomaterials, Drug DeliVery, Bionanotechnology and Molecular Recognition Laboratories, Department of Chemical Engineering and Department of Biomedical Engineering, UniVersity of Texas at Austin, UniVersity Code C0400, Austin, Texas 78712
Molecular imprinting is a promising technology that, although successfully used to recognize small molecules, has had many difficulties in recognizing macromolecules such as peptides and proteins. The current technologies used to achieve the macromolecular imprinting are incompatible with diagnosis and recognition in many life sciences applications such as medical devices, food additives, or drug delivery systems that require biocompatible products. We present here a new, biocompatible technology of protein imprinting by means of calcium alginate-based polymer capsules using ionic gelation and without toxic chemicals other than sodium alginate and calcium chloride. These molecular imprinting capsules are capable of recognizing higher quantities of protein than the existing technologies developed until now, with a simple formulation. Introduction Molecular imprinting polymers (MIPs) are cross-linked polymeric networks formed in the presence of a molecular mold or template. The subsequent release of the template allows the material to exhibit a selective “memory” with respect to the template. This, in turn, simulates the typical molecular recognition of biological systems. Molecular imprinting is a promising technology that has been successfully used to recognize small molecules1 such as herbicides, metal ions, and amino acids. Thus, the imprinting polymers can be used as sensors, chromatography beds, resins for separation processes, and analytical tools as in enzyme-linked immunosorbent assays (ELISA).1 However, the development of MIPs capable of recognizing large molecular weight biological molecules such as peptides and proteins has faced many difficulties,2 and the literature on this new field is not extensive.1 The current approach to macromolecular compound imprinting involves the inclusion of a template within a polymer formed from functional monomers and cross-linking agents. However, these technologies have not been compatible with diagnosis and recognition in life sciences applications such as medical devices, food additives, or drug delivery systems that require nontoxic, noncarcinogenic, biocompatible products. We believe that the system presented here, a system based on alginates as imprinting carriers, will provide a new and superior approach of molecular recognition. Alginates are water-soluble linear polysaccharides derived from brown algae and composed of alternating blocks of 1-4 linked R-L-guluronic and β-D-mannuronic acid residues. Alginates have excellent biocompability and biodegradability.3 An important property of these alginates is their capacity to form gels by reaction with divalent cations such as calcium or barium by binding between guluronic acid blocks in alginate and the divalent cations.3 For example, sodium alginate has hydroxyl and carbonyl groups that allow dipole/dipole interactions and hydrogen bonding between the alginate and the imprinting templates. Then, after the cross-linking process between sodium * To whom correspondence should be addressed. E-mail: peppas@ che.utexas.edu. Tel.: 001 (512)-471-6644. Fax: 001 (512)-471-8227. † University of Salamanca. ‡ University of Texas at Austin.
alginate and Ca2+ ions, protein molecules can be trapped inside the network and can be subjected to template removal and protein rebinding studies.4 Although the biocompability and biodegradability of alginates have been widely documented, only limited literature reports the use of alginate capsules to achieve the macromolecular imprinting. Regrettably, all studies up to now use the inverse suspension method that involves the use of organic chemicals such as chloroform and hexane that are incompatible with medical and alimentary purposes. The work of Zhang et al.5 was the first attempt at macromolecular imprinting using calcium alginate-based microcapsules via inverse suspension method. They improved the performance of the calcium alginate hydrogels using a small quantity of hydroxyethyl cellulose (HEC), thus achieving a recognition of 0.46 mg of bovine serum albumin (BSA) per gram of capsules. Zhao et al.2 used a hybrid of calcium phosphate/alginate-based microcapsules via inverse suspension method, which is an improvement of the protein macromolecularly imprinted calcium alginate capsules and achieves recognition of 0.66 mg of BSA per gram of alginate capsules. In this work, we present a new technology of protein imprinting based on calcium alginate polymer microcapsules via ionic gelation with no added chemicals other than sodium alginate and calcium chloride in order to produce carriers for biomedical applications for diagnosis of diseases and for detection in the food industry. Beyond being rapid and facile, the method can produce uniform pores with readily controlled size; in addition, the capsule size is controlled. The preparation process is easily carried out and takes little time compared with other traditional polymerization methods. Materials and Methods Materials. Sodium alginate from Macrocystis pyrifera (medium viscosity) was purchased from Sigma Chemicals; calcium chloride dehydrate, OmniPur, reagent grade, was purchased from EMD Chemicals; tris base (white crystals or crystalline powder), molecular biology, was purchased from Fisher BioReagentes, Fisher Scientific; and hydrochloric acid 1 N was purchased from Fisher Scientific. Methods. BSA Embedded Molecular Imprinted Calcium Alginate Capsules with Controlled pH. A specified amount of
10.1021/ie101068z 2010 American Chemical Society Published on Web 08/19/2010
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Table 1. Values of Volume/Concentration Used in the Calibration Process V, µL
1500
1200
1050
900
750
675
600
525
450
375
345
300
conc., mg/mL V, µL conc., mg/mL
10.0000 255 1.7000
8.0000 225 1.5000
7.0000 187.50 1.2500
6.0000 93.75 0.6250
5.0000 46.88 0.3125
4.5000 23.44 0.1563
4.0000 11.72 0.0782
3.5000 5.86 0.0391
3.0000 2.93 0.0195
2.5000 1.47 0.0098
2.3000 0.73 0.0049
2.0000
bovine serum albumin (BSA) was dissolved in deionized (DI) water (with pH adjusted to 4.2 by hydrochloride solution) up a concentration of 10 mg BSA/mL. Then, a specified amount of sodium alginate powder was added in the BSA solution until a concentration of 2 wt % corresponding to a viscosity of 2023 cP. To generate the imprinted capsules (MIPs), 3 mL of the alginate/BSA solution was added dropwise, through a needle using a sterile syringe, into the gelation solution, a 2 wt % calcium chloride aqueous solution, under stirring. This technique generated capsules with a particle size that ranged between 2 and 3 mm. It was possible to control the particle size by varying the liquid viscosity; by increasing this parameter, it is possible to increase the particle size diameter.3 The beads formed were kept in the cross-linking solution for 2 min. The microbeads were collected by filtration with a nylon filter membrane of 0.2 µm. A sample of the supernatant was taken and analyzed to measure the loss in the whole process of the generation of the capsules. To know the exact weight of the capsules, the syringe was weighed before and after the generation of the capsules. After the process, the beads were placed in a reactor for carrying out the procedure of releasing the protein. Also, capsules without the protein (BSA) were prepared for nonimprinted (NIP) and control samples. The procedure was the same as above. For the production of the NIP and control capsules, a specific amount of sodium alginate powder was dissolved into DI water to form a 2 wt % solution. Process of Template Removal. The previously generated MIPs were placed in a beaker containing the elution solution, a mixture of 1.0 wt % CaCl2 and tris buffer solution (0.05 M, pH of 7.4), which was prepared by dissolving a specific amount of calcium chloride powder in a 0.05 M tris buffer aqueous solution up to a concentration of 1% wt %. The pH of the buffer solution was set at 7.4 by adding HCl 1 N. The protein-removal process consisted of a series of cycles of agitation and extraction by means of a continuous system of renewal of the elution solution. After the removal process of the templates, the capsules were stored in the fridge for a few days in DI water to allow the swelling process of the capsules. In addition to producing the swelling process, these days also allowed the release of the remaining amount of template. Every day, the DI water was changed by filtration, and the supernatant was analyzed to know the release of the remaining amount of template. The NIP and control capsules were subject to the same process to maintain the same conditions as for the MIP capsules. Process of Protein Recognition. In order to achieve the recognition process, an accurately weighed amount of wet imprinted capsules (using filter paper to absorb the surface water) was placed in a centrifuge tube with 40 mL of a 1 mg/ mL BSA aqueous solution. The concentration of protein was evaluated with time by absorbance at 280 nm using a spectrophotometer. A calibration absorbance/concentration was carried out to know the quantity of protein released and absorbed in the removal and recognition processes, respectively, by means of spectrophotometer measurements. Calibration was based on 20 mL of a 10 mg/mL protein solution as the initial solution. Different volumes (Table 1) were taken from the initial solution,
to obtain different known concentrations of protein, and were disposed in micro-centrifuge tubes, and all volumes were filled with DI water until 1.5 mL. The detection was continued until a change in concentration of the solution was undetectable, and the equilibrium rebinding capacity was obtained. The process was repeated with the microcapsules without the protein, NIP, and the control capsules. The procedure was the same as above. The results were compared for the MIP and NIP capsules, with it being possible to know the amount of protein recognized. The control capsules were used to know the absorbance of the alginate with time. For experiments with control capsules, capsules without protein were placed in just 40 mL of DI water. Results and Discussion In this work, we developed novel alginate-based recognitive systems by using unique recognitive characteristics of such carriers. Indeed, sodium alginate has hydroxyl and carbonyl groups that allow dipole/dipole interaction and hydrogen bonding between alginate and the templates. To achieve good templated capsules, it is necessary to bind the negatively charged carbonyl groups of alginate on the template. Then, it is necessary to work below the isoelectric point of BSA (pI ) 4.7) to allow the BSA to behave as positively charged and favor a stronger attractive electrostatic interaction between the BSA (positively charged) and the alginate (negatively charged). To do that, the BSA solution was prepared with pH adjusted to 4.2 by hydrochloride solution. All previous research on protein imprinting based on alginate capsules used the inverse suspension method that involves the use of organic chemicals such as chloroform and hexane, which are incompatible with medical and alimentary purposes. However, the technology of protein imprinting based on calcium alginate polymer capsules developed in this work, via ionic gelation, with no more added chemicals than sodium alginate and calcium chloride, could be the future of the imprinting technology to the new life-based applications. Furthermore, this technology can save much time, in regard to the preparation of capsules, since the preparation time decreased significantly with respect to the traditional polymerization methods. Template Removal. In order to remove the template from the capsules, it was necessary to break the bond between BSA molecules and the alginate chains. To do this, the capsules were washed using an elution solution that is a mixture of 1.0 wt % CaCl2 and tris buffer solution (0.05 M, pH of 7.4). This pH forces the BSA to behave as negatively charged molecules, thus reducing the interactions between BSA and the negatively charged matrix of alginate. Also, the calcium of the elution solution interacts with the template and forces the template to diffuse out the capsules.6 After the removal process of the templates, the capsules were stored in DI water in the fridge for 2-3 days to allow the swelling process of the capsules. This process will permit the recovery of the original size of the capsules in the rebinding process, because the process of removal of protein causes the thickness of the membrane of the capsules to increase and therefore reduces the interior space inside the capsules, reducing
Ind. Eng. Chem. Res., Vol. 49, No. 20, 2010
Figure 1. Partial removal of template (90.04%) in only 5 h by mixing by combining discontinuous (agitation) and continuous (extraction) procedures. Note that reducing the time of washing with calcium solution prevents the thickening of the membrane of the capsules. The removal process was carried out in the following steps: (1) timing ) 3 h 4 min/continuous ) 1 h 42 min; (2) mixing ) 2 h 30 min/continuous ) 20 min; (3) mixing ) 35 min/continuous ) 9 min; (4) filtering; (5) stored ) 1 day; (6) stored ) 1 day.
Figure 2. Partial removal of template (87.11%) in only 5 h by combining discontinuous (agitation) and continuous (extraction) procedures. Note that reducing the time of washing with calcium solution prevents the thickening of the membrane of the capsules. The removal process was carried out in the following steps: (1) mixing ) 2 h 28 min/continuous ) 30 min; (2) mixing ) 1 h 40 min/continuous ) 10 min; (3) mixing ) 40 min/continuous ) 8 min; (4) filtering; (5) stored ) 1 day; (6) stored ) 1 day.
the space available inside the capsules to accommodate the protein again in the rebinding process. In the research work on protein imprinting based on alginate capsules, the procedure for removing BSA was completed in 48 h through successive washes and agitation, which is a discontinuous procedure. However, in the present research work, it is possible to achieve 87-90% release of the protein in only 5 h by combining discontinuous (agitation) and continuous (extraction) procedures (Figures 1 and 2). It is important to be able to reduce the time of release of the protein from 48 to 5 h, because during this process the capsules are washed with the elution solution composed of calcium. Because of the mechanism of formation of capsules by ionic gelation, the membrane of the capsules grows from outside to inside by diffusion of cations of calcium throughout the membrane. Although total protein release from the capsules was achieved (Figure 3), the process lasted >5 h, because it was necessary to use three cycles of mixing-extraction, with the last taking place overnight. Clearly, this was undesirable, because the calcium of the elution solution rendered the capsule membrane thicker with time. After the removal process, there is not available enough space inside the capsules to achieve the rebinding
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Figure 3. Total removal of template using overnight mixing. Although it achieved a total protein release from capsules because of the overnight mixing, it is undesirable because this process makes the capsule membrane thicker, leaving no space inside the capsules to achieve the rebinding process. The removal process was carried out in the following steps: (1) mixing ) 2 h 30 min/continuous ) 30 min; (2) mixing ) 4 h 30 min/continuous ) 22 min; (3) mixing ) overnight (12 h 48 min)/continuous ) 13 min.
Figure 4. Total removal of template without the overnight mixing, using 5 days of storage in DI water, producing the swelling process and allowing the release of the remaining amount of template without using calcium. The removal process was carried out in the following steps: (1) mixing ) 3 h 4 min/continuous ) 1 h 42 min; (2) mixing ) 2 h 30 min/continuous ) 20 min; (3) stored ) 3 day; (4) stored ) 1 day; (5) stored ) 1 day.
process. One solution to this problem is to do just two cycles of mixing and maintain the capsules in DI water for 5 days, renewing the water every day by filtering (Figure 4), during which the swelling process is produced as well as release of the remaining amount of template, without the presence of calcium. Anyway, as shown later in the recognition data, it is not necessary to get the total release of the template to attain satisfactory recognition results. Therefore, it is preferable to use the removal process as in Figures 1 and 2, which saves time and allows enough space in the inside of the capsules for the later rebinding process. Protein Recognition. In order to perform the recognition studies successfully, it was necessary to take into account that, since the calcium alginate capsules are biodegradable, they were going to disintegrate over time. Thus, filtration of the supernatant was required before considering final results of absorbance. Also, it was necessary to take into account that the alginate absorbs at 280 nm,7 and then the alginate absorption has to be followed by means of control capsules. The molecularly imprinted capsules presented in this work are capable of recognizing higher quantities of protein than the existing technologies developed until now, with a simple
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Ind. Eng. Chem. Res., Vol. 49, No. 20, 2010
Figure 5. Recognition studies corresponding to the partial removal process of Figure 1. In this figure, it is possible to observe a successful recognition of protein up to 1.5 mg of BSA per gram of capsules, compared to 0.7 mg achieved until now with existing techniques.
Figure 6. Recognition studies corresponding to the partial removal process of Figure 2. In this figure, it is possible to observe a successful recognition of protein up to 3.0 mg of BSA per gram of capsules, compared to 0.7 mg achieved until now with existing techniques.
formulation and with no more added chemicals than sodium alginate and calcium chloride. Zhang et al.5 achieve recognition of 0.46 mg of bovine serum albumin (BSA) per gram of capsules by using inverse suspension method to the generation of the capsules and hydroxyethyl cellulose (HEC) to improve the performance of the recognition. Zhao et al.2 uses a hybrid of calcium phosphate/alginate via inverse suspension method, achieving recognition of 0.66 mg of BSA per gram of capsules. This work is the first attempt at macromolecular imprinting using calcium alginate-based polymer capsules via ionic gelation method with no more added chemicals than sodium alginate and calcium chloride, allowing recognition that ranged between 1.5 and 3.0 mg of BSA per gram of capsules (Figures 5 and 6), corresponding to the template removal processes from Figures 1 and 2, respectively. Conclusions In this work, we have developed a new and biocompatible technique of protein imprinting by means of calcium alginatebased polymer capsules via ionic gelation that solves the difficulties encountered so far to achieve the recognition of macromolecules, such as proteins, being compatible with life sciences applications. The technique presented here is able to recognize up to 3.0 mg of BSA per gram of capsules compared to 0.7 mg achieved up to date with existing techniques developed until now, with a simple formulation. This is the first attempt of macromolecular imprinting by means of alginate, without the use of organic chemicals, that allows for use for medical and alimentary purposes.
Acknowledgment This work was supported in part by a grant from the Pratt Foundation. Literature Cited (1) Kimhi, O.; Bianco-Peled, H. Study of the interactions between protein-imprinted hydrogels and their templates. Langmuir 2007, 23, 6329– 6335. (2) Zhao, K.; Cheng, G.; Huang, J.; Ying, X. Rebinding and recognition properties of protein-macromolecularly imprinted calcium phosphate/alginate hybrid polymer microspheres. React. Funct. Polym. 2008, 68, 732–741. (3) Herrero, E. P.; Del Valle, E. M.; Galan, M. A. Development of a new technology for the production of microcapsules based on atomization processes. Chem. Eng. J. 2006, 117, 137–142. (4) Luzinov, I. Molecularly imprinted fibers with recognition capability. National Textile Center annual report; NTC Project: C05/CL01; National Textile Center: Blue Bell, PA, 2008. (5) Zhang, F. J.; Cheng, G. X.; Ying, X. G. Emulsion and macromolecules templated alginate based polymer microspheres. React. Funct. Polym. 2006, 66, 712–719. (6) Liu, Y.; Hunziker, E. B.; Randall, N. X.; De Groot, K.; Layrolle, P. Proteins incorporated into biomimetically prepared calcium phosphate coatings modulate their mechanical strength and dissolution rate. Biomaterials 2003, 24, 65–70. (7) Yu, Y.; Chen, S.; Bian, C.; Chen, W.; Xue, G. Facile synthesis of polyaniline-sodium alginate nanofibers. Langmuir 2006, 22, 3899–3905.
ReceiVed for reView May 10, 2010 ReVised manuscript receiVed August 2, 2010 Accepted August 6, 2010 IE101068Z
