Communication pubs.acs.org/Biomac
Preparation of Lipase-Coated, Stabilized, Hydrophobic Magnetic Particles for Reversible Conjugation of Biomacromolecules Marzia Marciello,†,‡ Juan M. Bolivar,†,§ Marco Filice,† Cesar Mateo,*,† and Jose M. Guisan*,† †
Instituto de Catálisis, CSIC, C/Marie Curie 2, Campus UAM, 28049 Madrid, Spain Instituto de Ciencia de Materiales, CSIC, C/Sor Juana Inés de la Cruz, 3, Campus UAM, 28049 Madrid, Spain § Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petergasse 12/I, 8010, Graz, Austria ‡
S Supporting Information *
ABSTRACT: This Communication presents the development of a novel strategy for the easy conjugation of biomolecules to hydrophobic magnetic microparticles via reversible coating with previously functionalized lipase molecules. First, the ability of lipase to be strongly adsorbed onto hydrophobic surfaces was exploited for the stabilization of microparticles in aqueous medium by the creation of a hydrophilic surface. Second, the surface amino acids of lipase can be tailored to suit biomolecule conjugation. This approach has been demonstrated by aminoepoxy activation of lipase, enabling the conjugation of different biomolecules to the magnetic particle’s surface. For example, it was possible to immobilize 70% of Escherichia coli proteins on the recovered particles. Furthermore, this strategy could be extended to other lipase chemical modification protocols, enabling fine control of biomolecule coupling. These conjugation techniques constitute a modular methodology that also permits the recycling of the magnetic carrier following use.
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Two strategies based on different protocols have been developed to conjugate biomolecules on the magnetic particle surface. The first strategy immobilizes biomolecules on the particle surface using irreversible covalent bonds. The advantages of covalent immobilization undoubtedly rely on the stabilization and irreversible anchoring of the target biomolecule to its magnetic carrier. On the other hand, irreversible covalent bonding prevents recycling of the magnetic particle when the biomolecule finishes its life cycle. For example, it is impossible to remove a covalently attached antibody from its magnetic carrier after it has lost its recognition ability. Another immobilization strategy is the physical adsorption (ionic or hydrophobic) of the biomolecules to the particle surface. Due to the possibility of biomolecule desorption, the ionic adsorption strategy presents some limitations when the particle−bioconjugate complex must be used in aqueous saline solutions (i.e., biologic fluids). Hydrophobic adsorption, on the other hand, could improve conjugate stability in aqueous solution. In fact, the interaction between two different hydrophobic surfaces is strengthened in an aqueous saline environment, drastically reducing the likelihood of biomolecule desorption. Hence, magnetic particles with hydrophobic coatings (hydrophobic polymers such as
agnetic nano- and microparticles are carriers with two main properties. They contain a magnetic core, allowing their separation from the reaction medium during processing, employment and recovery stages, and they have a large specific surface area, enabling a high load of target molecules.1,2 In recent years, small magnetic particles have received increasing attention in various fields, including biomedical and environmental applications, due to their size, high specific surface area, and low toxicity.3,4 For these reasons, they have been widely used for many different purposes, including intracellular uptake and separation,5 drug delivery,6 hyperthermia,7 magnetic resonance imaging contrast enhancement,8 enzyme and protein immobilization,9 protein purification10 and wastewater treatment.11 Among these applications, a very interesting field is the use of magnetic nanoparticles as carriers for biomolecules. Commonly, the binding of bioactive substances, such as enzymes, proteins, or antibodies, is accomplished through surface adsorption, covalent bonding, cross-linking with bifunctional reagents, gel-phase inclusion, or encapsulation.12 Many types of nano- and microparticles are available with various different coatings. Usually, a hydrophilic surface is considered to be better for the conjugation of biomacromolecules due to its compatibility with the hydrophilic surfaces of most target molecules.13 For example, hydrophilic particles have been used to investigate the impact of nanoparticle− protein interactions on biological responses at the nano scale, both in vitro and in vivo.14 © 2013 American Chemical Society
Received: October 25, 2012 Revised: February 1, 2013 Published: February 13, 2013 602
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Scheme 1. Proposed General Strategy for Lipase-Mediated Immobilization of Biomacromolecules on Hydrophobic Magnetic Particlesa
a
Hydrophobic magnetic particles coated with chemically modified lipases were used as a recyclable support for immobilization of biomacromolecules in water.
both a hydrophobic and hydrophilic moiety to coat hydrophobic magnetic particles and enable subsequent bioconjugation. Furthermore, due to its reversible noncovalent, hydrophobic immobilization, this straightforward strategy permits the recovery of unmodified magnetic particles for reuse in another cycle of modification−bioconjugation. With these features in mind and considering the many advantages of the solid-phase chemical modification of proteins, we developed the following particle-coating strategy. We first performed the chemical functionalization of the lipase enzymes while they were adsorbed on a solid support. We then removed them from the solid support and adsorbed them to surface of the hydrophobic magnetic particles (Scheme 1). To achieve this goal, octyl-sepharose was chosen as the support for the chemical modification of the enzyme surface due to its hydrophobic nature, easy handling during surface modification (simple filtration and washing are sufficient to eliminate excess reagent) and its porous surface that prevents particle aggregation caused by possible cross-linking due to the use of bi- or multifunctional reagents (epichlorohydrin, glutaraldehyde, diamines, etc.). Furthermore, its ability to selectively immobilize lipase from a mixture of different proteins (leading to a quantitative “one-pot” purificationimmobilization step) has been described.21 As a representative example, this work was performed using the commercially available Rhizomucor miehei lipase (RML). This lipase has a molecular weight of 33 KDa, many amino acid moieties available for chemical modification, and a lid composed of 14 primarily hydrophobic amino acids (SSSIRNWIADLTFV) (Figure 1a).22 RML has been previously immobilized by hydrophobic adsorption on octyl-sepharose, loading approximately 1.5 mg of pure lipase per gram of support. Once adsorbed, the carboxy residues of glutamic and aspartic acid on the protein surface were modified with ethylenediamine via amide formation using carbodiimide chemistry (Scheme 2a). In this way, new reactive alkylamine anchor points were created on the hydrophilic surface of the lipase. To verify the success of the amidation reaction, a picrylsulfonic acid (2,4,6-trinitrobenzenesulfonic acid, TNBS) colorimetric assay was performed. As is shown in Scheme 2b, compared to unmodified immobilized lipase, aminated lipase produced a significant color response, confirming the successful
polystyrene or latex) represent a good compromise between mechanical stability and the binding possibilities of the surface.4−9 The application of the magnetic particle/hydrophobic coating technology is associated with two significant challenges. First, to form a stable suspension in aqueous medium to prevent particle aggregation, hydrophobic particles are usually dispersed in the presence of surfactants. These detergents in many cases are not biocompatible with the final application and must be removed, thereby causing particle aggregation.4 Second, the local hydrophobic environment on the particle surface might interfere with the functionality of the attached target biomolecule. To avoid this problem, one could coat the particles with hydrophilic molecules, masking the particle’s hydrophobic character. The modification of hydrophobic surfaces for further functionalization and biomolecule conjugation is an active field requiring further study. For example, the binding of nonspecific proteins to the bead surface has been explored,15 but the use of specific proteins with high affinities for hydrophobic surfaces is an interesting alternative. The immobilization of lipases on magnetic particles by physical adsorption or via covalent binding has been described previously, but always with the final aim of exploiting their catalytic properties in organic synthesis or for the kinetic resolution of racemic mixtures.16 Lipases are enzymes that possess a hydrophilic surface together with a highly hydrophobic zone surrounding the catalytic site entrance (known as the lid).17 This hydrophobic zone allows the adsorption of the enzyme onto the surface of hydrophobic particles by an exclusive mechanism known as interfacial adsorption.18 The use of a functionalized lipase enzyme as a two-faced Janus molecule (Janus bifrons is the Roman god of beginnings and transitions are represented with a double-faced head),19 combining a highly hydrophobic zone with an affinity for magnetic particles together with a functionalized hydrophilic surface enabling conjugation with biomolecules, is a challenge. Lipases can be easily modified with different functional groups through chemical modification of the diverse amino acids residues on their surface and desorbed from the hydrophobic surface with surfactants.20 Hence, in this Communication, we propose a novel, elegant, and versatile methodology involving the use of lipase acting as 603
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epoxy arms on the lipase surface (Scheme 2b: 2 vs 3). To crosscheck the second chemical modification, the newly generated epoxy groups were first incubated with ethylenediamine and subsequently titrated with TNBS. The color intensity increased to the same level as that observed for aminated, immobilized lipase (Scheme 2b: 4 vs 2). It is worth noting that all these modifications were performed in aqueous, heterogeneous mixtures, permitting easy handling and recovery of the modified lipase. After functionalization with amino-epoxy groups, the lipases were desorbed from the octyl-sepharose support using sucrose laurate, a surfactant that can be easily removed using immobilized lipase hydrolysis.24 Specifically, a 0.5% w/v solution of sucrose laurate was used, and yields of approximately 40% were achieved. Subsequently, as previously described, the lipase solution was treated with a Thermomyces lanuginosus lipase derivative and dialyzed to remove the detergent. Upon completion of the pure modified lipase in aqueous solution (M-RML), immobilization onto hydrophobic, magnetic microparticles was studied (Scheme 3). Following removal of surfactant from the commercial suspension, 125 mg of polystyrene-coated, commercial magnetic microparticles were resuspended in a solution containing 2 mg of pure MRML in NaH2PO4 (5 mM) buffer with a pH of 7. Following immobilization (verified using the Bradford assay of the supernatant), approximately 1.25 mg of the modified lipase were immobilized on the microparticles (M-RML@MP) (10 mg per 1 g of magnetic microparticles), demonstrating that the region close to the lid was unaltered during the process. As previously outlined, the lipase layer coating the hydrophobic magnetic microparticles was expected to prevent or minimize particle aggregation in aqueous medium after removal of the surfactant. To investigate this, the aggregation of MRML-coated and uncoated polystyrene particles in the absence
Figure 1. Lipase surface analysis. Amino acids constituting the lid (green) and side-chain amino acidic residues that can be chemically modified. Red indicates carboxy groups from aspartic and glutamic acid, and blue indicates amino groups from lysine. (a) Rhizomucor miehei lipase (RML) (PDB file: 3tgl). (b) Geobacillus thermocatenulatus lipase (BTL) (PDB file: 2w22).
completion of the reaction (Scheme 2b: 1 vs 2). Subsequently, both the chemically inserted and the naturally present (due to the lysine residues) primary amine groups were modified with epichlorohydrin (Scheme 2b). 23 As a result of these modifications, a double reactive arm consisting of two secondary amine groups (capable of promoting ion exchange reactions) together with an epoxy group (able to covalently react with the nucleophilic groups of previously adsorbed molecules) was generated on the lipase surface. In this second step, the successful epoxidation of the aminated lipase surface was again checked using the TNBS colorimetric assay. In this case, the color intensity of the epoxidated lipase derivative dramatically decreased, confirming the presence of the amino-
Scheme 2. (a) Solid-Phase Chemical Modification of Lipase Adsorbed on Octyl-Sepharosea and (b) TNBS Assay of the AminoEpoxidation Reaction Stepsb
a
(Step 1) Chemical amination of surface carboxy groups. (Step 2) Epoxidation of amino groups of lipase surface (RML or BTL). b(1) Lipase adsorbed on octyl-sepharose (OS-RML). (2) Aminated derivative (OS-RML-NH2). (3) Epoxidation of derivative 2 (OS-RML-NH2-Epox). (4) The derivative described in 2 following reaction with ethylenediamine (OS-RML-NH2-Ep-NH2). 604
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Crude protein extracts from E. coli (a mixture of proteins with very different sizes and reactivities) were tested as model biomolecules. Prior to their immobilization, these proteins were moderately labeled with fluorescein isothiocyanate (FITC-Ps). To favor the preliminary anion exchange process, the immobilization reaction was first performed at pH 7 and low ionic strength (Scheme 4). Under these conditions, approximately 70% of the labeled protein (0.1 mg) was adsorbed onto 125 mg of M-RML@MPs, as was determined using the Bradford assay. To test the reversible ionic adsorption of the FTIC-Ps after a 30 min reaction, the microparticles were recovered and incubated for 30 min in an aqueous solution of 0.2 M NaCl. More than 85% (as determined by Bradford Assay of the supernatant) of the previously adsorbed FTICconjugated proteins were desorbed from the support, confirming the reversibility of the first step of this immobilization strategy. To promote the multipoint covalent immobilization of the previously adsorbed FTIC-Ps, the pH of the suspension proceeding from the first step was increased to 8.5, and the microparticle suspension was incubated for 15 h. To verify the covalent, irreversible immobilization of the FTICproteins, the chimeric magnetic microparticles (FTIC-Ps/MRML@MPs) were incubated in 0.2 M NaCl aqueous solution. Negligible desorption was observed, confirming the covalent immobilization of FTIC-Ps and the presence of epoxy groups on the lipase surface. Finally, to render the surface of the protein-lipase conjugate inert by blocking unreacted epoxy groups, the FTIC-Ps/M-RML@MPs were recovered, washed with distilled water, and incubated in 3 M aspartic acid solution at pH 8.5 for 15 h (Scheme 4). To further demonstrate this successful reaction, the FTICPs/M-RML@MPs chimeras were first washed with 0.2 M NaCl and then analyzed by fluorescence microscopy (FM). The resulting micrographs clearly confirmed the presence of the FTIC-labeled proteins on the microparticles surface (Figure 3a vs Figure 3b). Another interesting feature of this strategy is the possibility of recycling these modified particles after the operational life of the biomolecules on their surface is completed or reduced. In fact, as confirmed by the total disappearance of fluorescence as observed in FM analysis after incubation with 1% w/v sucrose laurate solution, the outer layer of the FTIC-Ps/M-RML@MPs chimera (the lipase-biomolecule conjugate) was easily removed, allowing the recovery and reuse of the magnetic microparticles in a new modification cycle (Figure 3c). To demonstrate the versatility of this strategy, the same experimental procedure was followed with the noncommercial
Scheme 3. Adsorption of Amino-Epoxidated Lipase on Polystyrene Magnetic Particles
of surfactants was studied using scanning electron microscopy (SEM). As clearly shown by micrographs in Figure 2, MRML@MPs have formed fewer aggregates than the uncoated MPs (Figure 2a vs 2b). Furthermore, the dynamic light scattering (DLS) analysis of M-RML@MPs and uncoated MPs confirmed that the former have a lower aggregation tendency and higher size uniformity (average hydrodynamic size: 2.2 ± 0.4 μm vs 3.3 ± 1.4 μm for M-RML@MPs and MPs, respectively) (Figure 2c). Once we demonstrated particle stabilization resulting from the lipase coating, the ability of such derivatives to surface conjugate biomolecules was studied. The modified, lipase-coated magnetic microparticles were expected to conjugate to a variety of different drugs and biomolecules with a wide range of molecular weights and chemical reactive groups (i.e., protein, antibody, DNA, RNA etc.) with a two-step mechanism (Scheme 4).23 In the first rapid step, by performing the reaction at a pH where the target biomolecule possesses a net negative charge, ionic adsorption of the target to the positively charged secondary amino groups coating the lipase surface is promoted. A covalent intramolecular reaction between the nucleophilic groups of the target and the epoxy groups of the lipase can then occur. The unreacted epoxy groups were then blocked with aspartic acid to render the surface inert to further reactions. Beyond the unreacted epoxy groups blocking, the reaction with aspartic acid also confers a strongly net negative charge on the particle surface. As a result, the unspecific adsorption of undesired proteins (most of them negatively charged at neutral pH) and the aggregation of the particles themselves are hampered.
Figure 2. Aggregation analyses of lipase-coated and uncoated hydrophobic magnetic particles: (a) SEM micrographs of polystyrene magnetic particles in buffered aqueous medium. (b) SEM micrographs of polystyrene magnetic particles coated with modified lipase in buffered aqueous medium. (c) DLS analysis of lipase-coated (M-RML@MPs) and uncoated (MPs) hydrophobic magnetic particles. 605
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Scheme 4. Covalent Immobilization of FTIC-Labeled Proteins on ML@MP by a Two-Step Mechanisma
a
(a) Ionic adsorption. (b) Subsequent intramolecular covalent immobilization. (c) Unreacted epoxy groups blocking.
Figure 3. Fluorescence micrographs: (a) Polystyrene magnetic particles coated with modified RML (M-RML@MPs) (the slight fluorescent background is due to the natural fluorescence of the lipase). (b) FTIC-proteins covalently linked to amino-epoxidated lipase coated particles (FTICPs/M-RML@MPs). (c) polystyrene magnetic particles after chimer desorption (MPs).
Geobacillus thermocatenulatus lipase (BTL) (Figure 1b).25 Similar results were achieved with this enzyme, including successful amino-epoxidation of the enzyme surface, 45% yield for desorption with 0.5% w/v sucrose laurate solution, the immobilization of 1.1 mg of modified BTL on 125 mg of hydrophobic magnetic microparticles, the successful covalent immobilization of FTIC-Ps through the two-steps mechanism, and successful desorption of the FTIC-Ps/M-BTL chimers from the MP surface (see the Supporting Information for more details). This series of experiments confirmed the modularity of the proposed strategy. In conclusion, we describe here a novel and versatile strategy to stabilize hydrophobic particles in aqueous medium and then reversibly conjugate biomolecules to their surface. This strategy consists of a solid phase chemical modification of the hydrophilic face of lipase enzymes followed by the subsequent immobilization of these proteins on the hydrophobic magnetic particles through their hydrophobic faces (Janus character). The versatility of this method lies in the use of different lipase sources (either commercial or not, owing to their peculiar interfacial activation mechanism), a wide variety of possible chemical modifications to the lipase surface (depending on the desired conjugation protocol), and a broad variety of different drugs and biomolecules (i.e., protein, antibody, DNA, RNA, etc.). Using this straightforward strategy, biosensors or carriers for drug targeting and gene therapy could be easily manufactured following lipase inactivation by site-directed
mutagenesis of the catalytic serine or the use of cholinesterase inhibitors.4 Furthermore, due to the fine control of the biomolecule-to-particle coupling chemistry, this strategy could be a useful tool for understanding nanoparticle−protein interactions in both in vitro and in vivo biological systems.15 Finally, this modular strategy also permits the easy recycling of hydrophobic magnetic particles at the end of the life-cycle of their conjugated biomolecules. This feature is a very important characteristic considering the high overall cost of such particles when compared with other commonly used reagents. Additional characteristics of this versatile immobilization strategy will be demonstrated in subsequent papers concerning the conjugation of different small molecules or biomacromolecules for various biotechnological purposes.
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ASSOCIATED CONTENT
S Supporting Information *
Experimental procedures are described in the Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] (C.M.);
[email protected] (J.M.G.). Fax: +34-915854760. Phone: +34-915854809. 606
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work has been sponsored by the Spanish Ministry of Science and Innovation (Intramural projects (2008801058)). REFERENCES
(1) Pan, Y.; Du, X.; Zhao, F.; Xu, B. Chem. Soc. Rev. 2012, 41, 2912− 2942. (2) Singamaneni, S.; Bliznyuk, V. N.; Binek, C.; Tsymbal, E. Y. J. Mater. Chem. 2011, 21, 16819−16845. (3) Peteiro-Cartelle, J.; Rodríguez-Pedreira, M.; Zhang, F.; Gil, P. R.; Del Mercato, L. L.; Parak, W. J. Nanomedicine 2009, 4, 967−979. (4) Yang, H-W; Hua, M-Y; Liu, H-L; Huang, C-Y; Wei, K-C Nanotechnol., Sci., Appl. 2012, 5, 73−86. (5) Shubayev, V. I.; Pisanic, T. R., 2nd; Jin, S. Adv. Drug Deliver. Rev. 2009, 61, 467−477. (6) Mahon, E.; Salvati, A.; Baldelli Bombelli, F.; Lynch, I.; Dawson, K. A. J. Controlled Release 2012, 161, 164−174. (7) Guardia, P.; Di Corato, R.; Lartigue, L.; Wilhelm, C.; Espinosa, A.; Garcia-Hernandez, M.; Gazeau, F.; Manna, L.; Pellegrino, T. ACS Nano 2012, 6, 3080−3091. (8) Peng, X. H.; Qian, X.; Mao, H.; Wang, A. Y.; Chen, Z. G.; Nie, S.; Shin, D. M. Int. J. Nanomed. 2008, 3, 311−321. (9) Ansari, S. A.; Husain, Q. Biotechnol. Adv. 2012, 30, 512−523. (10) Zhou, L.; Wu, J.; Zhang, H.; Kang, Y.; Guo, J.; Zhang, C.; Yuan, J.; Xing, X. J. Mater. Chem. 2012, 22, 6813−6818. (11) Wang, F.; Hu, Y.; Guo, C.; Huang, W.; Liu, C. Z. Bioresour. Technol. 2012, 110, 120−124. (12) Mukhopadhyay, K.; Sutradhar, S.; Modak, S.; Roy, S. K.; Chakrabarti, P. K. J. Phys. Chem. C 2012, 116, 4948−4956. (13) Mateo, C.; Palomo, J. M.; Fernandez-Lorente, G.; Guisan, J. M.; Fernandez-Lafuente, R. Enzyme Microb. Technol. 2007, 40, 1451−63. (14) Monopoli, M. P.; Åberg, C.; Salvati, A.; Dawson, K. A. Nat. Nanotechnol. 2012, 7, 779−786. (15) Goli, K. K.; Rojas, O. J.; Ozçam, A. E.; Genzer, J. Biomacromolecules 2012, 13, 1371−1382. (16) Marciello, M.; Filice, M.; Palomo, J. M. Catal. Sci. Technol. 2012, 2, 1531−1543. (17) Smith, L. C.; Faustinella, F.; Chan, L. Curr. Opin. Struct. Biol. 1992, 2, 490−496. (18) Verger, R. Trends Biotechnol. 1997, 15, 32−38. (19) Ropponen, J.; Nummelin, S.; Rissanen, K. Org. Lett. 2004, 6, 2495−2497. (20) Fernandez-Lorente, G.; Godoy, C. A.; Mendes, A. A.; LopezGallego, F.; Grazu, V.; de Las Rivas, B.; Palomo, J. M.; Hermoso, J.; Fernandez-Lafuente, R.; Guisan, J. M. Biomacromolecules 2008, 9, 2553−2561. (21) Bastida, A.; Sabuquillo, P.; Armisen, P.; Fernández-Lafuente, R.; Huguet, J.; Guisán, J. M. Biotechnol. Bioeng. 1998, 58, 486−493. (22) Brady, L.; Brzozowski, A. M.; Derewenda, Z. S.; Dodson, E. J.; Dodson, G. G.; Tolley, S. P.; Turkenburg, J. P.; Christiansen, L.; HugeJensen, B.; Norskov, L.; Thim, L. Acta Crystallogr. B 1992, 48, 307− 319. (23) Mateo, C.; Fernández-Lorente, G.; Abian, O.; FernándezLafuente, R.; Guisán, J. M. Biomacromolecules 2000, 1, 739−745. (24) Marciello, M.; Mateo, C.; Guisan, J. M. Colloid. Surface. B 2011, 84, 556−560. (25) Carrasco-López, C.; Godoy, C.; De Las Rivas, B.; FernándezLorente, G.; Palomo, J. M.; Guisán, J. M.; Fernández-Lafuente, R.; Martínez-Ripoll, M.; Hermoso, J. A. Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 2008, 64, 1043−1045.
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