Enzymatic Nanocomposites with Radio Frequency Field-Modulated

Letter Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

pubs.acs.org/journal/abseba

Enzymatic Nanocomposites with Radio Frequency Field-Modulated Activity Yulia I. Andreeva,† Andrey S. Drozdov,*,† David Avnir,‡ and Vladimir V. Vinogradov*,† †

ACS Biomater. Sci. Eng. Downloaded from pubs.acs.org by CALIFORNIA STATE UNIV FRESNO on 11/29/18. For personal use only.

Laboratory of Solution Chemistry of Advanced Materials and Technologies, ITMO University, St. Petersburg 197101, Russian Federation ‡ Institute of Chemistry and the Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel S Supporting Information *

ABSTRACT: The control over enzymatic activity by physical stimuli is of interest to many applications in medicine, biotechnology, synthetic biology, and nanobionics. Although the main focus has been on optically responsive systems, alternative strategies to modulate the enzymatic activity of hybrid systems are needed. Here we describe a radiofrequency (RF) field controlled catalytic activity of an enzymatic sol−gel composite. Specifically, the activity of bovine carbonic anhydrase entrapped in sol−gel-derived magnetite (enzyme@ ferria) composite was accelerated by a factor of 460% compared to its initial value, by applying the RF field of 937 A/m, with fast response time. This acceleration is reversible and its magnitude controllable. An acceleration mechanism, based on RF-induced heating of the magnetite by the Néel relaxation effect, is proposed and proven. The entrapment within a sol−gel matrix solves the problem of enhancing activity by heating without denaturing the enzyme. RF-controlled enzymatic composites can be potentially applied as biological RF sensors or to control biochemical reactions within living organisms. KEYWORDS: enzymatic nanocomposites, enzymatic activity modulation, electromagnetic field, stimuli-controlled biocomposite, sol−gel magnetic field on magnetic nanosuspensions;22,23 and the heat acceleration of a thermophilic enzymes entrapped in a polymer containing magnetite nanoparticles by applying an alternating magnetic field.24,25 In this article, we present the first example of activity enchantment of a nonthermopilic enzyme, carbonic anhydrase, under the influence of a RF field. The observed effect was achieved through the entrapment of enzymes within magnetite sol−gel matrix and was the combined result of two phenomena: the heating of magnetite nanoparticles under the influence of RF-fields by the Néel relaxation effect,26 and the high thermal stability of the enzymes entrapped within sol−gel derived materials.27−30 Specifically, we have selected carbonic anhydrase from bovine erythrocytes (CAB) for that study, one of the most efficient and well-studied and a member of an important class of enzymes used in Alzheimer’s disease treatment.31 Upon the irradiation of this biocomposite by the RF field at a frequency of 210 kHz and amplitude of 937 A/m, the catalytic reaction rate was boosted up to 460% of its initial value in a fast and reversible manner. Entrapment of enzymes in magnetite sol−gel materialsa long-standing challenge  became possible after the develop-

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timuli-responsive enzyme−material composites are of central interest for a wide variety of biomedical and biotechnological needs. The enzymatic activity of such materials typically is modulated by external stimuli that are chemical in nature, such as varying the pH1,2 or introducing specific ions3,4 or cofactor molecules.5,6 Alternatively, it can be physical in nature, such as varying the temperature7,8 or triggering/modulating with a variety of irradiations, including ultrasound,9 light,10,11 electric fields,12−14 or magnetic fields.15,16 The application of radio frequency (RF) emission for modulating enzymatic activity, the focus of this report, is attractive because of its excellent penetrability which still leaves tissues unharmed, and yet compared with the other stimuli listed above, research on RF applications has been much less explored. Examples include enzymes entrapped in a thermoresponsive polymeric gel doped with Fe2O3 nanoparticles (NPs), where an RF field was used to change a polymer conformation, allowing the enzyme to interact with substrate;17,18 the entrapment of enzymes within a polymeric matrix with magnetic rods, such that low-frequency caused them to vibrate, increasing the mass transfer within the polymer;19 enzymes immobilized in chitosan beads with magnetic NPs, allowing a rotating magnetic field to increase the reaction rate due to the induced magnetic stirring;20,21 changing the enzymatic reaction rate by applying a nonheating © XXXX American Chemical Society

Received: July 23, 2018 Accepted: October 30, 2018 Published: October 30, 2018 A

DOI: 10.1021/acsbiomaterials.8b00838 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Letter

ACS Biomaterials Science & Engineering

Figure 1. CAB@ferria nanocomposites structure. (a) SEM image of the CAB@ferria particles. (b) EDX mapping of the CAB@ferria particles, iron signal corresponds to magnetite, nitrogen signal corresponds to CAB. (c) HR-SEM image of the CAB@ferria composite. (d) TEM image of the CAB@ferria, the crystalline interspacing of magnetite is shown in the inset; (e) XRD spectra of CAB@ferria (correlated to JCPDS No. 19−0629 shown as red lines). (f) Schematic representation of the entrapment of the enzyme within the interstitial porosity of the aggregated ferria.

(JCPDS No. 19−0629) with the typical main peak at 35.45° attributed to the plane with Miller indices of (311) (Figure 1e). Also, a typical 2.86 Å crystalline spacing of magnetite is seen in the HR-TEM image (Figure 1d inset).34 For composite materials with mass fractions of CAB below 10% wt, full entrapment was observed, and enzyme release into buffer solutions was absent. While the physical entrapment of enzyme within sol−gel matrix prevents the leaching of the enzyme, it was still catalytically active, with in-diffusion of substrate molecules and out-diffusion of product molecules through the pore network (Figure 1f). The activity of the CAB@ferria composite was evaluated by measuring the rate of p-nitrophenyl acetate (pNPA) hydrolysis to p-nitrophenol. In the absence of RF irradiation, the hydrolysis rate of pNPA at 37 °C by was found to be 23 μmol/min per mg of the composite. To evaluate the effect of RF field on the enzymatic activity the biocomposite was placed inside an inductive RF coil, and an alternating magnetic field with a frequency of 210 kHz and field amplitude of 937 A/m was applied under thermostatic conditions at 37 °C. Under the influence of the RF field, a significant boost of the catalytic activity to 105 μmol/min was observed, which is 457% increase of its original value (Figure 2a). It can be seen from the kinetics curve that the reaction rate rapidly reaches a steady mode and remains literally on the same level during the whole experiment. The reversibility of this acceleration was demonstrated by following the response to switching the RF field on and off in the following experiment: A cuvette with a buffered dispersion of the CAB@ferria particles was subjected to several cycles of RF-irradiation (210 kHz, 937 A/m) and relaxation periods of 2 min each. The catalytic activity of the CAB@ferria composite under the influence of RF-field (210 kHz, 937 A/m) was repeatedly enhanced to an average of 450% of its initial rate while returning to its original value when the field was removed (Figure 2b).

ment of a stable magnetite sol (ferria) with a mean particle diameter of 10 nm, free of any sol-stabilizing agents, and at a neutral pH.32 The unique feature of the ferria sol is its high zeta potential (valued +31 mV at pH 7.0) ensuring the high colloidal stability under biocompatible conditions and the capability to undergo sol−gel transition at the room temperature.33 These features originate from the chemical properties and composition of the ferria NPs: because of special synthetic conditions the NPs surface is enriched by Fe(II)−OH groups, which, on one hand shift the isoelectric point of the NPs to higher pH values, and on the other, facilitate the formation of interparticle Fe−O−Fe bonds in a course of partial dehydration of the surface at the room temperature, leading to a mesoporous inorganic matrix formation (see Figure S1).32 The enzyme was entrapped within a magnetite sol−gel derived matrix, by a recent protocol we have developed, described in ref 34. Buffered CAB solution was added to the ferria hydrosol with a subsequent water removal and sol−gel condensation process. The produced hybrid ceramic material was washed with buffer solution to remove any adsorbed enzyme molecules and grinded to obtain a composite mesoporous powder (grain size 5−7 μm, Figure 1a) designated as CAB@ferria (see Materials and Methods for the details). The composite material demonstrated a high degree of homogeneity with the uniform distribution of the components as is seen from EDX analysis (Figure 1b). The mesoporosity of the composite material gave rise to a nitrogenaccessible surface area of 120 m2/g and average pore diameters of 8.2 nm, as calculated by the BET and BJH equations, respectively (Figure S2), with the enzyme, entrapped and buried within the material (Figure 1c, d). The elementary building blocks of the aggregated powder were highly crystalline magnetite NPs with a mean diameter of about 10 nm, as revealed by XRD and TEM. The XRD pattern of the ferria nanoparticles correspond to the pattern of magnetite B


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slower rate, and the temperature reached 64 °C only after 16 min of irradiation (Figure 3d). Although the measured temperature and the visualized heat images were taken from the surface of the sample, the temperature within the material should be on the same level due to the short relaxation time of the heat conduction within magnetite nanoparticles (≈10 ns),38 resulting in homogeneous heating of the entrapped distributed enzyme molecules in the CAB@ferria composite. It should be noted that no change of the dispersing water temperature was detected during the whole experiment, due to the low volume (5 × 10−4 %) and mass (2 × 10−3 %) fractions of the magnetic material, and it was maintained at the level of 37 °C. At the same time it is known that the temperature difference for the surface of magnetically heated nanoparticles and the media can reach large values (up to 70 °C for 5 nm),39 so it can be assumed that while the water temperature was remained at the same level, the CAB@ferria NPs was heated to the temperatures which is close to the one measured on the air. The next question to be answered is, how did the entrapped enzyme react to the temperature rise? In contrast to all the systems described earlier, free CAB is known to have low thermal stability and readily denaturates at 68 °C.40 This fact is in agreement with the experimental results of free CAB measured by dynamic scanning calorimetry (DSC) (Figure 4a). In contrast, entrapment within the magnetite sol−gel matrix enhanced the thermal stability of CAB and shifted its denaturation temperature up to 97 °C, as seen from the DSC curve (Figure 4a). The origin of this stabilization comes from the strong interactions of the enzyme molecules with the pores of the sol−gel derived matrix, and were described earlier for this kind of materials.1,41,42 Because of the higher thermal stability of entrapped CAB, it was capable of performing the enzymatic reactions at higher temperatures and even demonstrated an Arrhenius-like behavior on a broader

Figure 2. (a) Kinetics of pNPP formation by CAB@ferria with and without the applied RF-field; (b) catalytic activity of CAB@ferria with periods of irradiation and relaxation are 2 min. The experiments were carried out under thermostatic conditions.

For the interpretation of the observed phenomena, the influence of the RF-field on CAB@ferria is to be addressed. It is known, that magnetic materials are generating heat under the influence of high-frequency oscillating magnetic fields as a result of relaxation processes, which involves the gradual alignment of the magnetic moments during the magnetization process. These relaxation processes may mainly take place through two distinct mechanisms: the Brownian relaxation and the Néel relaxation, but for NPs with diameters around 10 nm (our case) the Néel mechanism is thought to be predominant.35 In that case, heat is dissipated from the magnetic particles by the delay in the relaxation of the magnetic moment through the rotation within the particle: This effect is frequently used for biomedical purposes for so-called magnetic hypothermia of cancer tissues.36,37 To measure the heating effect of the RF field on CAB@ferria, the material was investigated with a heat imager (Figure 3). Heat image of the irradiated material in air showed that the composite is heated up to 62 °C under the influence of the RF field within the 1 min of exposure (Figure 3b). Further heating occurs at a much

Figure 3. Temperature maps of CAB@magnetite subjected to RF fields: (a) field is off; after (b) 1, (c) 5, and (d) 16 min of irradiation. The temperature was measured at M2 and HS1 marks in addition to the heat map. C


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Figure 4. (a) DSC curves of CAB@ferria and free CAB; (b) temperature−activity dependence of free CAB and CAB@ferria. All experiments were carried out under thermostatic conditions.

Da was calculated to be 6.7 at 20 °C indicating that the catalytic process was affected but not controlled by diffusion limitations (see the Supporting Information for full calculations). In order to better understand the difference between free CAB and CAB@ferria composite kinetic parameters Vmax and KM were measured at 20 and 50 °C, and the results are presented in Table 1 (Figure S3). It was found that for both systems the kinetic parameters are temperature-dependent: both systems were stable in the selected temperature region and demonstrated the rise of Vmax with temperature elevation. It can also be noted, that CAB@ferria composite showed approximately two folds higher value of KM and 1.5 folds larger Damkö hler number, which means an increase in the contribution of diffusion limitations to the kinetics of the overall reaction as the reaction rate grows faster than the diffusion rates. To prove the authenticity of the RF field effect on the enzymatic activity, we performed several control experiments. First, the possible effect of an applied field with an amplitude of 937 A/m on the activity of free CAB was measured in solution, with or without dispersed magnetite: no acceleration of the reaction was observed under these conditions (see Figures S4 and S5). Second, CAB was entrapped within a nonmagnetic matrix, alumina boehmite, according to the procedure of ref 26 and then subjected to the RF field under similar conditions, again showing no acceleration of the enzymatic activity (Figure S6). Third, the importance of the 3D entrapment was demonstrated by comparing with adsorption of CAB on the surface of the magnetite particles: slight inactivation to 90% of the initial activity was observed (Figure S7), reflecting the lower thermal stability of the adsorbed enzymes in contrast with the entrapped one.11 Fourth, if indeed we are witnessing thermally induced RF acceleration combined with matrix protection from denaturation, then this observation should not be unique for a CAB. Indeed, for confirmation purposes of the CAB study, a potential RF induced acceleration was checked for acid phosphatase AcP@ferria: exposure to the same RF field (210 kHz, 937 A/m) led to acceleration of the reaction rate by a factor of 3, from 51 to 157 μmol/min (Figure S8). In summary, we described a new class of stimuli-responsive biocomposites, based on enzymes entrapped within sol−gelderived magnetite. We showed that the enzymatic activity of the sol−gel composites is enhanced under the influence of a RF field in a fast and reversible manner as an outcome of the following effects: heating of the CAB@ferria composite by the Néel relaxation mechanism, and thermal stabilization of the enzyme entrapped within sol−gel derived magnetite. The proof-of-concept was demonstrated for CAB and AcP, but it can be expected that the same principle can be applied for other enzymes. RF-sensitive enzymatic systems can be an

temperature range compared with the free enzyme (Figure 4b). The pNPA hydrolysis rate by CAB@ferria increased until it reached its maximum near 60 °C (111 μmol min−1 mg−1), whereas for the free enzyme, a significant drop of the activity after 40 °C was observed. The experiments were carried out under thermostatic conditions with preliminary incubation at the experimental temperature, so that the rate of the reaction was constant during the whole experiment. It is notable that the catalytic activity of CAB@ferria at 60 °C is close to the one measured under the influence of the RF field (111 and 105 μmol min−1 mg−1, respectively). This fact can be used as an indirect indicator of the irradiated composite temperature in water solution. It should be noted that the rate of the reaction per milligram of the enzyme is 45-fold lower for the entrapped CAB in comparison with the free enzyme (18 vs 817 μmol min−1 mg−1 for entrapped and free CAB, respectively). This difference can be attributed to diffusional limitations of the reaction carried out by the heterogeneous catalyst. The effective diffusion coefficient Deff of pNPA within sol−gel matrix was estimated to be 38 folds lower than in solution valued at 1.8 × 10−7 and 7 × 10−6 cm2 s−1, respectively (see the Supporting Information for calculations). To determine whether the reaction is controlled primarily by the diffusion of the substrate or by the catalytic ability of the immobilized enzyme the Damköhler number was evaluated using eq 1: Da =

Vmax Deff K δ M

(1)

where Vmax is the maximal reaction rate, Deff is the effective diffusion coefficient, δ is the effective thickness of the unstirred layer through which the substrate must diffuse, and KM is Michaelis−Menten constant (Table 1, see the Supporting Table 1. Kinetic Parameters of the Enzymatic Systems System CAB, 20 °C CAB, 50 °C CAB@ferria, 20 °C CAB@ferria, 50 °C

KM (μmol/L)

Vmax (μmol/(min mg))

Damköhler number

14.93 30.3 38.46

1785 5181 153

6.7

69.9

775

9.9

Information for full calculations). The Damköhler number is the dimensionless ratio of reaction velocity to transport velocity, determining whether diffusion rates or reaction rates are more “important” for defining a steady-state chemical distribution over the length and time scales of interest. For Da ≫ 1, the reaction rate is much faster than the diffusion rate distribution is said to be diffusion limited, whereas when 1 ≫ Da, diffusion is much faster than the reaction.43 In ous system D


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(15) Zakharchenko, A.; Guz, N.; Laradji, A. M.; Katz, E.; Minko, S. Nature Catalysis 2018, 1, 73. (16) Bakshi, S. F.; Guz, N.; Zakharchenko, A.; Deng, H.; Tumanov, A. V.; Woodworth, C. D.; Minko, S.; Kolpashchikov, D. M.; Katz, E. Nanoscale 2018, 10, 1356−1365. (17) Seino, S.; Yoshida, K.; Shikakura, T.; Watanabe, K.; Koga, Y.; Nakagawa, T.; Yamamoto, T. A. J. Magn. Soc. Jpn. 2014, 38, 98−101. (18) Liu, T.-Y.; Hu, S.-H.; Liu, K.-H.; Shaiu, R.-S.; Liu, D.-M.; Chen, S.-Y. Langmuir 2008, 24, 13306−13311. (19) Amstad, E.; Kohlbrecher, J.; Muller, E.; Schweizer, T.; Textor, M.; Reimhult, E. Nano Lett. 2011, 11, 1664−1670. (20) Thomas, C. R.; Ferris, D. P.; Lee, J.-H.; Choi, E.; Cho, M. H.; Kim, E. S.; Stoddart, J. F.; Shin, J.-S.; Cheon, J.; Zink, J. I. J. Am. Chem. Soc. 2010, 132, 10623−10625. (21) Timin, A. S.; Gao, H.; Voronin, D. V.; Gorin, D. A.; Sukhorukov, G. B. Adv. Mater. Interfaces 2017, 4, 1600338. (22) Klyachko, N. L.; Sokolsky-Papkov, M.; Pothayee, N.; Efremova, M. V.; Gulin, D. A.; Pothayee, N.; Kuznetsov, A. A.; Majouga, A. G.; Riffle, J. S.; Golovin, Y. I.; Kabanov, A. V. Changing the Enzyme Reaction Rate in Magnetic Nanosuspensions by a Non-Heating Magnetic Field. Angew. Chem., Int. Ed. 2012, 51, 12016−12019. (23) Efremova, M. V.; Veselov, M. M.; Barulin, A. V.; Gribanovsky, S. L.; Le-Deygen, I. M.; Uporov, I. V.; Kudryashova, E. V.; SokolskyPapkov, M.; Majouga, A. G.; Golovin, Y. I.; et al. In Situ Observation of Chymotrypsin Catalytic Activity Change Actuated by Nonheating Low-Frequency Magnetic Field. ACS Nano 2018, 12, 3190. (24) Suzuki, M.; Aki, A.; Mizuki, T.; Maekawa, T.; Usami, R.; Morimoto, H. Encouragement of Enzyme Reaction Utilizing Heat Generation from Ferromagnetic Particles Subjected to an AC Magnetic Field. PLoS One 2015, 10, No. e0127673. (25) Suzuki, M.; Hayashi, H.; Mizuki, T.; Maekawa, T.; Morimoto, H. Biochemistry and biophysics reports 2016, 8, 360−364. (26) Fannin, P. J. Phys. D: Appl. Phys. 1991, 24, 76. (27) Avnir, D.; Coradin, T.; Lev, O.; Livage, J. J. Mater. Chem. 2006, 16, 1013−1030. (28) Chapurina, Y. E.; Drozdov, A. S.; Popov, I.; Vinogradov, V. V.; Dudanov, I. P.; Vinogradov, V. V. J. Mater. Chem. B 2016, 4, 5921− 5928. (29) Shabanova, E. M.; Drozdov, A. S.; Ivanovski, V.; Suvorova, I. I.; Vinogradov, V. V. RSC Adv. 2016, 6, 84354−84362. (30) Shabanova, E. M.; Drozdov, A. S.; Fakhardo, A. F.; Dudanov, I. P.; Kovalchuk, M. S.; Vinogradov, V. V. Sci. Rep. 2018, 8, 233. (31) Scozzafava, A.; Mastrolorenzo, A.; Supuran, C. T. Expert Opin. Ther. Pat. 2006, 16, 1627−1664. (32) Drozdov, A. S.; Ivanovski, V.; Avnir, D.; Vinogradov, V. V. J. Colloid Interface Sci. 2016, 468, 307−312. (33) Shapovalova, O. E.; Drozdov, A. S.; Bryushkova, E. A.; Morozov, M. I.; Vinogradov, V. V. Arabian J. Chem. 2018, DOI: 10.1016/j.arabjc.2018.02.011. (34) Anastasova, E. I.; Ivanovski, V.; Fakhardo, A. F.; Lepeshkin, A. I.; Omar, S.; Drozdov, A. S.; Vinogradov, V. V. Soft Matter 2017, 13, 8651−8660. (35) Rosensweig, R. E. J. Magn. Magn. Mater. 2002, 252, 370−374. (36) Perigo, E. A.; Hemery, G.; Sandre, O.; Ortega, D.; Garaio, E.; Plazaola, F.; Teran, F. J. Appl. Phys. Rev. 2015, 2, No. 041302. (37) Jordan, A.; Scholz, R.; Wust, P.; Fähling, H.; Felix, R. J. Magn. Magn. Mater. 1999, 201, 413−419. (38) Jayhooni, S.; Rahimpour, M. Superlattices Microstruct. 2013, 58, 205−217. (39) Riedinger, A.; Guardia, P.; Curcio, A.; Garcia, M. A.; Cingolani, R.; Manna, L.; Pellegrino, T. Nano Lett. 2013, 13, 2399−2406. (40) McCoy, L. F., Jr; Wong, K.-P. Biochemistry 1981, 20, 3062− 3067. (41) Drozdov, A. S.; Shapovalova, O. E.; Ivanovski, V.; Avnir, D.; Vinogradov, V. V. Chem. Mater. 2016, 28, 2248−2253. (42) Avnir, D.; Braun, S.; Lev, O.; Ottolenghi, M. Chem. Mater. 1994, 6, 1605−1614. (43) Mears, D. E. Ind. Eng. Chem. Process Des. Dev. 1971, 10, 541− 547.

attractive tool for creation of biochemical radio receivers, radio-controlled catalytic materials, or on-demand operating biochemical systems. Due to the fact that the RF-generated heat is rapidly dissipating in water, acceleration of biochemical reactions can be unpaired from heating of the media, affecting only a few nanometers of the surrounding area. In contrast with light radiation, RF fields have better penetration into biological media and are not inclined to damage biological objects. Such systems can be potentially applied for the augmentation of living organisms such as a bacteria, cells, or even an animals, and can be applied to the control of biochemical processes.44

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.8b00838. Materials and Methods section, supporting calculations and additional figures related to materials characterization and enzymatic kinetic evaluation (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Andrey S. Drozdov: 0000-0001-7260-3095 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Russian Science Foundation, grant 18-79-00266. REFERENCES

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Enzymatic Nanocomposites with Radio Frequency Field-Modulated

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