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Loading Capacity versus Enzyme Activity in Anisotropic and Spherical Calcium Carbonate Microparticles Senem Donatan,†,# Alexey Yashchenok,*,†,∥,# Nazimuddin Khan,‡,# Bogdan Parakhonskiy,*,§,∥,⊥,# Melissa Cocquyt,⊥ Bat-El Pinchasik,†,∇ Dmitry Khalenkow,⊥ Helmuth Möhwald,† Manfred Konrad,‡ and Andre Skirtach†,⊥ †

Department of Interfaces, Max Planck Institute of Colloids and Interfaces, Golm/Potsdam D-14476, Germany Enzyme Biochemistry Group, Max Planck Institute for Biophysical Chemistry, Göttingen D-37077, Germany § A.V. Shubnikov Institute of Crystallography RAS, 119333 Moscow, Russia ∥ Remote Controlled Theranostic Systems Lab, Institute of Nanostructres and Biosystems, Saratov State University, 410012 Saratov, Russia ⊥ Department of Molecular Biotechnology, NB-Photonics Group, Ghent University, Ghent 9000, Belgium ∇ Department of Physics at Interfaces, Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany

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‡

S Supporting Information *

ABSTRACT: A new method of fabrication of calcium carbonate microparticles of ellipsoidal, rhomboidal, and spherical geometries is reported by adjusting the relative concentration ratios of the initial salt solutions and/or the ethylene glycol content in the reaction medium. Morphology, porosity, crystallinity, and loading capacity of synthesized CaCO3 templates were characterized in detail. Particles harboring dextran or the enzyme guanylate kinase were obtained through encapsulation of these macromolecules using the layer-by-layer assembly technique to deposit positively and negatively charged polymers on these differently shaped CaCO3 templates and were characterized by confocal laser scanning fluorescence microscopy, fluorometric techniques, and enzyme activity measurements. The enzymatic activity, an important application of such porous particles and containers, has been analyzed in comparison with the loading capacity and geometry. Our results reveal that the particles’ shape influences morphology of particles and that, as a result, affects the activity of the encapsulated enzymes, in addition to the earlier reported influence on cellular uptake. These particles are promising candidates for efficient drug delivery due to their relatively high loading capacity, biocompatibility, and easy fabrication and handling. KEYWORDS: vaterite, enzyme, calcium carbonate, polyelectrolyte, enzyme-catalyzed reaction

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role in the uptake of drug carriers into cells.20 Capsules with cubical,21,22 semispherical,22 and elliptical23 shapes showed a relatively high penetration rate in cells in comparison with spherical capsules produced from the same material. For example, red blood cells elongate upon passing through small vessels or capillaries. But in the case of particles or even capsules, extensive compressibility is not possible. This prompts development of different anisotropic carriers. To date, anisotropic particles and carriers have been assembled by different methods,24−28 whereas fabrication of anisotropic calcium carbonate particles through controlling the ratio of salts also has been reported.23,29 Such different anisotropic shapes of drug delivery carriers were shown to facilitate cell uptake,37,25 i.e., the uptake of capsules by cells depended on the aspect ratio of particles and capsules.

INTRODUCTION A promising strategy to enhance the cellular targeting efficacy in drug delivery systems is mimicking the biological behavior,1 which is often related to shape-specific, anisotropic, and noncovalent interactions between biological molecules.2 In addition, the delivery of biomolecules can be enhanced by choosing a delivery vehicle of a desired geometry. Therefore, development of nonspherical carriers with elongated, filamentous morphologies,3 or anisotropic delivery systems4−8 is regarded as an essential advance due, in part, to more effective cellular targeting9,10 and uptake.11 Alteration of biological responses was reported for particles with similar compositions, but with different geometries12−14 entering the biodistribution pathways.15−17 Not only were capsules produced by various anisotropic shapes but they were also shown to change shape in the case of hydrogel-based containers.13,18,19 Inhalation is viewed as a particularly important area of medical application of anisotropic particles because of their favorable hydrodynamic properties. The shape has also been identified to play a major © 2016 American Chemical Society

Received: March 23, 2016 Accepted: May 11, 2016 Published: May 11, 2016 14284

DOI: 10.1021/acsami.6b03492 ACS Appl. Mater. Interfaces 2016, 8, 14284−14292

Research Article

ACS Applied Materials & Interfaces

conducted. Promising applications of such porous particles were recently identified in enzyme-catalyzed reactions.56−58 Here, we investigated the effectiveness of enzyme-catalyzed reactions in different types of particles, and note that in addition to their application as microcarriers in enzymology, such particles can also be used for a broad range of applications in drug delivery. In our studies presented here, we focus on cross-comparison of the loading efficiency versus the enzymatic activity, the property that has not yet been studied in depth.

Polymeric carriers, in addition to other common drug carriers, e.g., nano- and microparticles, liposomes and red blood cells, represent an important class of vehicles that are often designed for mimicking naturally occurring delivery systems. Polymeric capsules engineered through the highly versatile layer-by-layer (LbL) technique have attracted particular attention in the last decades, because they offer flexibility of design and multifunctionality, and they can be used in a broad range of drug delivery applications.30−32 The LbL technique is based on the consecutive adsorption of oppositely charged polyelectrolytes on a preformed charged template core that is chemically dissolved after the coating process. LbL capsules have already been widely used for encapsulation of biological substances especially enzymes like urease,33,34 luciferase,35 glucose oxidase, and peroxidase.33,36 Many therapeutics and biologically relevant materials can be introduced into LbL films via noncovalent interactions under physiological conditions. The biological compatibility of these materials does not change significantly during loading, these capsules are not cytotoxic and can be made biodegradable, which makes them suitable candidates for in vivo applications. As the LbL technology combines the versatility of manipulating the composition, surface chemistry, and dimensions of nanostructured thin films with the easy functionalization of diverse therapeutics and/or biomolecules, it provides a powerful tool for the nanoscale synthesis of novel drug delivery systems. LbL capsules are typically manufactured using various materials as core templates.37 In particular, CaCO3 particles represent a very important class of templates for LbLengineered capsules in drug delivery applications due to their highly porous structure, biocompatibility,38 degradability in mild conditions,39 and ease of fabrication.40 The shape of these CaCO3 templates is mainly spherical, and their crystal structure is usually vaterite. Among the three polymorphs (calcite, aragonite, or vaterite) of CaCO3, vaterite is thermodynamically the least stable one.41 However, for drug delivery applications, it is an attractive candidate, because vaterite particles are not toxic38 and highly porous capable of immobilizing many small39,42,43 and large molecules44−47 into their interior structure.48 In other application areas such as biomineralization or nanomaterial processing, CaCO3 particles are utilized in a diverse range of shapes because their crystallization mechanism can be easily altered, using organic additives.49−52 If additives such as dipeptides,52 polypeptides,49 peptide-type block copolymers, or inorganic materials, which bind to peptides,51 are added during precipitation of CaCO3 the shape and size of CaCO3 particles can be altered from nanorods to mesocrystals. On the other hand, organic additives can cause undesirable cytotoxic effects in drug delivery applications. Herein, we report on fabrication of new anisotropic LbL53,54 containers templated on spherical and nonspherical vaterite CaCO3 particles and study the influence of geometry and the number of polyelectrolyte multilayers on enzyme-catalyzed reactions in these particles. In this work, we have used a novel approach to control the shape of the particles. In addition to previously reported methods of controlling the shape, where the ratio of salts was used as the driving mechanism,29,55 we have extended the synthesis by adding polyethylene glycol together with changing the salt ratio: New shapes of particles, which have not been reported to date resulting from this approach. A detailed scanning electron microscopy (SEM) study of particle surfaces and confocal laser scanning microscopy (CLSM) studies of loaded molecules has been

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EXPERIMENTAL SECTION

Materials. Poly(allylamine hydrochloride) (PAH, Mw 70 kDa), poly(sodium 4-styrenesulfonate) (PSS, Mw 70 kDa), tetramethylrhodamine isothiocynate-dextran (TRITC-dext, Mw 70 kDa), ethylenediaminetetraacetic acid (EDTA), calcium chloride dihydrate (CaCl2·2 H2O), sodium carbonate (Na 2CO3), adenosine 5′triphosphate (ATP), guanosine 5′-monophosphate (GMP), pyruvate kinase, and lactate dehydrogenase were purchased from Sigma-Aldrich, Germany. Nicotinamide adenine dinucleotide reduced form (NADH) and phosphoenolpyruvate were from Roche Diagnostics GmbH (Mannheim, Germany). Ethylene glycol (EG) solution (99%) was purchased from Alfa Aesar (Karlsruhe, Germany). Recombinantly produced and purified human guanylate kinase (hGMPK) was prepared as reported previously.59 In all procedures, Milli-Q water was used with characteristic resistivity of 18 MOhm·cm. Synthesis of Calcium Carbonate Templates. Formation of colloidal particles from supersaturated solution (relative to that of CaCO3) was initiated by rapid mixing (600 rpm) of equal volumes (10 mL) of CaCl2 and Na2CO3 solutions with varying concentration ratios (1M:1M, 0.1M:1M, and 1M:0.1M) in the presence of different concentrations of EG solution (25%, 50%, and 80%). The mixture was constantly agitated on a magnetic stirrer at 600 rpm for 30, 60, and 90 min. The pH values of the initial salt solutions, Na2CO3 and CaCl2, were 9.5 and 7.4, respectively. After precipitation, the resulting particles were thoroughly washed three times with deionized water and 50% ethanol in order to remove residual EG and salts. The precipitate was dried at 70 °C for 1 h. The calcite particles were obtained via a 24 h immersion of 10 mg of vaterite particles in water. After incubation, the particles were collected by centrifugation and dried at 70 °C for 1 h. Polyelectrolyte Modification. Synthetic polyelectrolytes in solutions containing 2 mg/mL each of PAH and PSS and 0.5 M NaCl were subsequently adsorbed onto CaCO3 templates using the LbL deposition technique.60,61 For this, presynthesized CaCO3 particles (5 mg) were incubated with 2 mL of polyelectrolyte solution during 15 min. Then, particles were collected by centrifugation (3000 rpm, 3 min) and washed with distilled water. This procedure was repeated 3 times for each layer of polyelectrolyte. Characterization. Particle Morphology. Samples were prepared by applying a drop of the particle suspension to a glass slide followed by drying overnight. Scanning electron microscopy (SEM) images were recorded using a Philips XL30 electron microscope operating at the acceleration voltage of 3 kV. The axis ratio and particle sizes were calculated using the ImageJ program. In addition, the same software was used for estimating the number of particles from SEM images. Loading Capacity. Emission intensities of TRITC-dext-loaded CaCO3 particles were measured to estimate the loading capacity. 1 mL of 0.1 M TRITC-dext solution was added to 10 mg of presynthesized particles. After 30 min of incubation, the particles were precipitated by centrifugation (3 min, 3000 rpm). Optical absorption intensity of the supernatant was measured at 542 nm (absorption maximum of TRITC-dext). For estimation of the loading capacity in weight percentage, the loading amounts were normalized by the weight of dry calcium carbonate particles. The statistical analysis of the loading capacity was done by the one-way ANOVA test. The significant differences were defined at p < 0.05. For loading hGMPK onto CaCO3 particles, 1 mL of 45 μM (1 mg/ mL) hGMPK solution was added to 5 mg of presynthesized CaCO3 14285


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Figure 1. SEM images of calcium carbonate particles synthesized by stirring at 600 rpm for 30 min by mixing CaCl2 and Na2CO3 salts in 25% ethylene glycol (EG) (a−c), in 50% EG (d−f), and 83% EG (g−i) medium. The scale bars on images correspond to 2 μm.

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particles. After 30 min of incubation, the particles were isolated from the hGMPK solution and washed by centrifugation. The quantity of protein remaining in the supernatant was determined by the Bradford method measuring optical absorption intensity at 600 nm.62 For estimation of the loading capacity in weight percentage, the loading amounts were normalized by the weight of dry calcium carbonate particles. Crystallinity. Powder X-ray diffraction analysis was performed on a PDS120 diffractometer (Nonius GmbH, Solingen) with Cu Kα radiation, and the patterns were recorded between 20° and 60° (2θ) angles. Porosity. The surface area and the pore size distribution of CaCO3 microparticles were determined following the Brunauer−Emmett− Teller (BET) method of nitrogen adsorption/desorption at 77.35 K. The data were collected by an Instrument QuadraSorb Station 2, Version 5.04 (Quantachrome Instruments). ζ-Potential. The ζ-potentials of CaCO3 particles were measured in water suspension using a Malvern Zetasizer 4. Capsule Morphology. Confocal micrographs were taken with a Leica TCS SP confocal laser-scanning microscope (CLSM) (Leica, Germany) in inverted microscope mode, equipped with a 100×/1.4− 0.7-oil immersion objective. NADH-Dependent Spectroscopic Assay. The catalytic activity of human guanylate kinase (hGMPK) was determined by the standard NADH-dependent enzyme-coupled assay using a JASCO V-650 UV− vis spectrophotometer.59,62−67 The formation of ADP and GDP by hGMPK was coupled to two additional reactions catalyzed by pyruvate kinase (PK) and lactate dehydrogenase (LDH), respectively. As shown in the reaction scheme below (Figure 4a), each mole of phosphoryl group transferred from ATP produces two moles of NDPs, and consequently two moles of NADH are oxidized to NAD+.65 The timedependent absorbance change due to NADH oxidation was monitored spectrophotometrically at 340 nm because NADH absorbs light at 340 nm (ε = 6.22 mM−1 cm−1), whereas NAD+ does not. All measurements were performed at 25 °C in a buffer containing 100 mM Tris, pH 7.5, 100 mM KCl, and 10 mM MgCl2. In these assays, encapsulated hGMPK was used at 10 μL of enzyme-loaded particles from a suspension of 5 mg of the particles in a reaction volume of 1 mL.

RESULTS AND DISCUSSION Calcium carbonate microparticles were synthesized by vigorously mixing two highly soluble salts: calcium chloride and sodium carbonate. Mixing solutions with equal concentrations of these two salts leads to the formation of spherical particles,68 and this approach was widely used for production of templates or cores60 for polyelectrolyte multilayer microcapsules. Recently, a new approach was proposed in which nonequimolar concentrations in equal volumes of the corresponding salts were mixed.29,55 In such case, there is a limiting concentration of one salt relative to that of the other salt, leading to the formation of anisotropic particles. In the present study we have found that the initial salt concentration has a significant impact on the nucleation of calcium carbonate particles, so that even at equal but relatively high concentrations of both salts and through the addition of ethylene glycol anisotropic particles can be produced. To obtain stable vaterite particles, the working pH range was chosen between 9.0 and 9.5,55 with stirring at 600 rpm for 30 min, because calcium carbonate is more stable in this pH range. For synthesis of anisotropic CaCO3 particles, the initial calcium chloride and sodium carbonate salt concentrations were altered. We also performed particle synthesis in the presence of ethylene glycol, which permits for control of the reaction kinetics by slowing down the reaction. In this way, the size of the calcium carbonate particles was decreased from the micrometer to the submicrometer range.69 The surface morphology, crystallinity, porosity, and loading capacity of resulting CaCO3 particles were controlled in this work as a function of the reaction time, the concentration of the initial salt solutions, the solubility of salts, and mixing speed. Calcium carbonate microparticles in ellipsoidal, rhomboidal, and spherical geometries were produced by changing the concentrations of initial salt solutions at constant pH, mixing speed, and reaction time. The initial salt concentration ratio was found to play a major role in the formation of CaCO3 particles 14286


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ACS Applied Materials & Interfaces (Figure 1). The second parameter that we altered was the addition of EG to the reaction mixture while varying the concentrations. Because of the high viscosity of EG, it decreases the diffusion of ions, and at the same time reduces the solubility of CaCO3 crystals. In addition, EG prevents the recrystallization of CaCO3 polycrystals from the vaterite phase to calcite. In this study, we used different concentrations of EG (25, 50, and 80% in the reaction mixture). At low EG (25%) and relatively high salt concentrations (1 M), we obtained spherical particles, but all showed a strong tendency to aggregate (Figure 1a), most likely due to high amounts of salts present at the start of the reaction in a small local volume. Increasing the EG concentration, or decreasing the salt concentrations, proved to be beneficial to avoid such agglomeration while obtaining separate particles. At high, albeit identical, initial concentrations of salts (e.g., 1 M), spherical, elliptical, and rhomboidal CaCO3 particles were formed (Figure 1d,g). The axis ratio, also termed aspect ratio, of the fabricated structures varies between 1.68−1.75. With increasing EG concentration, the size of the long axis of the particles decreases from 2.1 to 1.5 μm For synthesis in nonequilibrium conditions, the concentration ratios of the salts were raised up to 10 (Figure 1b,e,h,c,f,i). In this case, the shape of the CaCO3 particles transforms to a star-like morphology (for the low EG concentration), and also to elongated distorted ellipsoidal forms if the concentration of calcium ions is ∼10 times lower than that of carbonate ions (Figure 1b,e). The aspect ratio of these particles lies in the narrow range between 2.70 and 2.93. Spherical particles were obtained when the concentration of carbonate ions was ∼10 times lower than that of calcium ions (Figure 1c,f). Increasing the EG content to 50%−80% in the reaction medium, leads to increased density that, in turn, reduces the stability of precipitated CaCO3 particles.41 This change led to the formation of ellipsoidal particles with different shapes (Figure 1d,g,h). The ellipsoidal CaCO3 particles shown in Figure 1g have aspect ratios of 1.8 to 1.9. Our findings reveal that solubility of salts is one of the key parameters for the shaping process of anisotropic CaCO3 particles. Our SEM analyses (Figure 1) show that a high concentration of carbonate ions leads to the formation of anisotropic rhomboidal or ellipsoidal geometries with different axis ratios (Figure 1d,e,g,h), whereas low concentration of carbonate results in creation of isotropic spherical particles (Figure 1f,i). These results are consistent with data obtained in our previous study55 In our current work, we optimized physicochemical parameters in order to control the morphology of anisotropic CaCO3 microparticles. However, we could not avoid secondary nucleation, which occurs on the side surfaces, resulting in irregular star-like or flower-like structures (Figure 1a,b,c,e). X-ray diffraction (XRD) combined with the ultramicrotomic cutting technique reveals the inner structure of the calcium carbonate particles. All types of particles that were synthesized by adjusting the ratio of salt solutions possess the porous structure shown in Figure 2. Indeed, the powder X-ray studies reveal the presence of vaterite morphology in all particles (Figure S1). As expected, the sizes of particles obtained at a higher percent of EG are smaller than those synthesized at a lower content of EG, and this is consistent with previous studies.13 Loading of the produced anisotropic particles is an important aspect for their application as carriers of small and large

Figure 2. (a) Loading capacity determined by measuring the absorbance of TRITC-dextran (70 kDa) molecules in anisotropic calcium carbonate microparticles. (b−d) Confocal fluorescence microscopy images of elliptical and spherical microparticles filled with TRITC-dextran. The scale bars in panels b and d images are 2 μm, and in panel c it is 10 μm.

molecules of biological and biomedical interest. We studied the loading capacity of TRITC-dextran-containing particles using absorption measurements to determine the concentrations of dextran sulfate, a model macromolecule, before and after loading. In addition, confocal laser scanning microscopy (CLSM) was used to confirm the presence of molecules inside the particles. Figure 2 shows that elliptical particles, or star-like particles had a comparable loading capacity of above 8 μg/mg, similar to spherical particles (Figure 2a). The loading capacity of elliptical particles with sizes of the long axis of 1.4 and 2.1 μm was lower, but still above 4 μg for 1 mg of CaCO3 particles. Typical CLSM images (Figure 2b−d) confirm the presence of TRITC-dextran molecules inside the particles. The vaterite structure of such particles can be preserved against formation of the calcite structure by polyelectrolyte coatings. This is an important step of practical applicability, because nonstabilized vaterite particles recrystallize into nonporous rhomboidalshaped particles. Figure 3 shows close-up SEM images of recrystallized CaCO3 particles having calcite crystalline structure (Figure 3a) and star-like (Figure 3b) calcium carbonate particles.

Figure 3. SEM images of (a) rhomboidal and (b) star-like shaped calcium carbonate particles. The scale bars correspond to 2 μm.

A particularly important application of CaCO3 particles is shown to be enzyme-catalyzed reactions.58 This is due to the fact that microparticles, or microcapsules templated on them, allow for a high loading capacity retaining enzymes inside the microcapsules and preserving their catalytic activity. For this reason, we have chosen five types of particles with different shapes: spherical large, spherical small, small elliptic, 14287


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Table 1. Particles with Different Shape and Size Were Used To Study Their Surface Area, Loading Capacity, and Activity of Loaded Enzymes particles shape rhomboidal small spherical small elliptical large spherical star-like

size [μm]

total surface area BET, ST [m2/g]

surface area, S [m2/g]

loading capacity, LC [%] 0.6 ± 0.3 4 ± 0.9

activity, A [μM/min·mg]

activity of loaded enzymes per mg of CaCO3 particles, [μM/min/mg particles]

control free enzyme activity, [μM/min·mg]

6.8 7

4 28

32 42

2.8 ± 0.9 0.6 ± 0.1

10 22

>0.8 6.3

± ± ± ±

30

3.5

3 ± 0.2

23

69

33

15−22

1.0

2.8 ± 2.3

28

78

49

18

>1.3

4.8 ± 0.9

31

148

37

0.8 0.8 1.4 3.6

0.2x 0.2x 0.3 0.7

4 ± 0.5

Figure 4. Catalytic activity of the human enzyme guanylate kinase (hGMPK) loaded on calcium carbonate particles of different shapes and surrounded by varying numbers of polyelectrolyte layers. (a) Scheme of the three-step enzyme-catalyzed reaction measuring hGMPK activity. This coupled-enzyme assay monitors hGMPK activity of the enzyme loaded on calcium carbonate microparticles of various shapes. The decrease in absorbance at 340 nm, associated with NADH oxidation in the third reaction step, is directly proportional to GMP phosphorylation in the first reaction. All measurements were performed at 25 °C in buffer A containing 100 mM Tris, pH 7.5, 100 mM KCl, and 10 mM MgCl2. The final reaction mixture contained 2 mM ATP, 1 mM GMP, concentration of the encapsulated or control hGMPK, which contained in 0.05 mg of vaterite particles (10 μL from 1 mL of 5 mg/mL particles), 0.5 mM PEP, 4 units of pyruvate kinase (PK), 0.25 mM NADH, and 5 units of lactate dehydrogenase (LDH) in 1 mL reaction volume. (b) Catalytic activities of the reaction shown by control free enzyme concentration corresponded to the particles of different loading capacity (black line) and particles loaded with enzyme (blue line) in (a) conducted in romboidal (■), small spherical (●), large spherical (○), star-like (★), and elliptical (◆) against the number of polyelectrolyte layers adsorbed on the surface of microparticles. U/mg is defined as μmol of substrate transformed into product per minute per milligram of protein under the given conditions of measurement.

To avoid desorption or enzyme release, activity studies were performed immediately after loading. All types of the particles are crystallographically stable in this time range, and desorption of the molecule was not more than 10%, which corresponds to the dynamic equilibrium of the adsorbed molecules in solution. The coupled assay measuring kinase activity consists of three enzymes and their substrates. Here, hGMPK is the enzyme of interest, whereas PK (pyruvate kinase) and LDH (lactate dehydrogenase) are helper enzymes (Figure 4a). Mixing all constituents of the assay without hGMPK (or microparticles) gives a constant absorbance (∼1.5 at 340 nm) due to NADH (0.25 mM). The addition of free hGMPK enzyme, or enzymeloaded microparticles, to the reaction mixture causes the oxidation of NADH to NAD+, and the optical absorption, which decreases at 340 nm, was followed in real time under steady state-kinetic conditions. The enzymatic activity is proportional to the loading capacity, but not all molecules contribute to the activity:

star-like, and as control nonporous rhomboidal calcite particles. All these types of particles have different surface areas (Table 1). Moreover, once covered with a polyelectrolyte shell, these particles or capsules permit small-molecule substrates and products to freely diffuse through the pores of the polymeric shell, while stabilizing in their interior the enzyme and protecting it from degradation. To study this reaction, we have subsequently measured the loading capacity as well activity of encapsulated human guanylate kinase (hGMPK) loaded on the produced different types of the particles. For particle loading capacity, two different factors play a role: size and shape. Depending on the particle type and on the ratio of external and internal surfaces, the protein, which is quite a big molecule, can immobilize in the pores, but it is mostly adsorbed on the external surface. Table 1 shows that among particles of similar sizes, calcite particles (rhomboidal) have the smallest surface area and they possess the smallest loading capacity. Because of their external surface, the star-like particles exhibited a better protein loading capacity in comparison to TRITC-dextran (which for almost all types of particles was less than 1%). Because the molecular weight of native hGMPK (23 kDa) is smaller than that of dextran (70 kDa), higher loading capacities are expected. This was confirmed by our results: The loading capacity of hGMPK ranges from 2 to ∼5 wt %.

activity ∼ LC × (Stotal − Sint passive)

(1)

where Stotal is the total surface area determined from BET, Sint passive is the internal surface area, which contributes to the total loading capacity, but does not contribute to the activity. 14288


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product molecules (here: ADP, GDP). As such, covering particles by polyelectrolyte multilayers is an effective method to control the enzymatic activity.

The activity of enzymes encapsulated in 1 mg of calcite (rhomboidal) particles is the lowest, consistent with the lowest loading capacity of these particles (Table 1). It is conspicuous that, although the loading capacity of star-like particles is only slightly higher, the activity of encapsulated enzymes (per 1 mg of particles) is significantly higher (Table 1). This finding can be assigned to the surface structure of these particles, since elliptical particles have higher surface area (Table 1), but lower activity. This is particularly pronounced for small particles, both spherical and elliptical. The dependence of resulting catalytic activities (U/mg) on the number of polyelectrolyte layers is presented in Figure 4b. Catalytic activity was determined for all types of particles: (i) without coating, (ii) with one layer of the negatively charged polyelectrolyte polystyrenesulfonate (PSS), and a subsequent layer of poly(allylamine hydrochloride) (PAH), and (iii) with two to four consecutively applied layers of these polymers. These polymers were chosen because they are well-studied and represent a suitable model system. Large spherical, elliptical, and star-shaped particles exhibited similar catalytic activities (Figure 4b). The highest activity was observed for noncoated particles. This is expected because of an easier access of the substrates ATP and GMP to, and release of the products ADP and GDP from, the enzyme guanylate kinase in such a case. It is worth noting that smaller spherical particles were somewhat less catalytically active than larger micrometer-sized particles discussed above. Despite the high loading capacity (4 w%), the vaterite particles have a similar pore size (35−45 nm) and almost 2 times higher ζ-potential (−20 mV versus −10−12 mV for large particles38,69) than the largest star-like particles or large spherical ones. For this reason, both of these factors can influence the final catalytic activity: Dense packing of the enzyme molecules could restrict their flexibility/activity, and charged substrates (ATP, GMP) and products (ADP, GDP) cannot freely diffuse into/out of small particles because of the charge. Rhomboid-shaped particles exhibited lowest activity, as expected, because of their largely nonporous nature. The catalytic activity was observed to decrease with the increasing number of polyelectrolyte layers, but the activity was still significant even after application of four layers (Figure 4b). Our study shows that particles of different shapes, and microcapsules built on such particles, can serve as microcarriers in a broad range of applications, including those that allow tuning of permeability and release;70 this can be achieved by varying the density of charges,71 or by the number of applied polyelectrolyte layers.72,73 The catalytic activity of enzymeloaded spherical and star-like particles decreases with the increasing number of layers (Figure 4). A decrease of activity by 4 times for small spherical particles, 3.5 times for star-like, and 2.5 times for large spherical particles already occurs with the presence of at least two layers of polyelectrolytes. We observed that the decrease of activity upon coating with polyelectrolyte layers is less pronounced for ellipse-like particles, i.e., upon coverage with four layers there still remains a relatively high enzymatic activity. Star-like particles have a larger roughness and surface area as compared to spherical and elliptical particles of comparable size. For the star-like particles, a large number of molecules are located on the surface without penetrating the particle. But for the elliptic particles, enzyme molecules enter inside the porous structure, and can slow down the release of the reaction

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CONCLUSIONS In this study, we have presented the fabrication of polyelectrolyte-coated carriers based on the vaterite-type of CaCO3 microparticles with rhomboidal, ellipsoidal, star-like, and spherical geometries. A novel approach has been developed for the fabrication of anisotropic shapes of stable vaterite CaCO3 particles by extending the range of the relative salt ratios, and also by the addition of ethylene glycol. Our results indicate that a high concentration of carbonate ions leads to the formation of anisotropic rhomboidal or ellipsoidal geometries with different aspect ratios, whereas a low concentration results in isotropic spherical particles. An increase of the ethylene glycol content in the reaction medium affects the aspect ratio, enhances the porosity, and, as a result, the protein loading capacity. Our analysis demonstrates that the shape of the particles has the influences on its morphology which, in turn, affects both the loading capacity and the catalytic activity, and that the enzymatic activity increases with an increase of the loading capacity of carbonate particles. In particular, star-like particles, although exhibiting only a slightly higher loading capacity, display a significantly higher enzymatic activity. It was found that the shape of particles influence their morphology, which, in turn, affects the activity of encapsulated enzymes. It was also found that the addition of polyelectrolyte multilayers slows down the kinetics of the catalytic activity. Thus, particles developed in this work are viewed as promising candidates for drug delivery applications by virtue of their high loading capacity, biocompatibility, and easy fabrication and handling, whereas data on the loading capacity and activity of enzymes encapsulated in these particles, as investigated in this work, should be useful for guiding further practical applications.

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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/acsami.6b03492. XRD data of spherical, ellipsoidal and rhomboidal CaCO3 particles (PDF).

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AUTHOR INFORMATION

Corresponding Authors

*Bogdan Parakhonskiy. E-mail: bogdan.parakhonskiy@ugent. be. *Alexey Yashchenok. E-mail: [email protected]. Author Contributions

# These authors contributed equally. The paper was written through contributions of all authors. All authors have given approval to the final version of the paper.

Funding

Government of the Russian Federation (grant no. 14.Z50.31.0004 to support scientific research projects implemented under the supervision of leading scientists at Russian institutions and Russian institutions of higher education), RFBR research project no. 15-29-01172, DAAD PhD scholarship, recurrent funding through the Max Planck Institute, BOF (Bijzonder Onderzoeksfonds) of the University of Ghent (Belgium) and FWO (Fonds Wetenschappelijk Onderzoek) of 14289


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Vlaanderen for support. We also thank the Marie-Curie IRSES project “DINaMIT” and ERA.Net Rus project “IntelBioComp”. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Rona Pitschke for SEM images, Ingrid Zenke for Xray diffraction measurements. S.D. thanks TUBITAK. A.M.Y thanks the Alexander von Humboldt-Stiftung. A.M.Y and B.V.P acknowledge the Government of the Russian Federation (grant no. 14.Z50.31.0004 to support scientific research projects implemented under the supervision of leading scientists at Russian institutions and Russian institutions of higher education), RFBR research project no. 15-29-01172 ofi_m. N.K. has been supported by a DAAD Ph.D. scholarship. M.K. acknowledges continued funding through the Max Planck Institute. A.G.S. acknowledges support of BOF (Bijzonder Onderzoeksfonds) of the University of Ghent (Belgium) and FWO (Fonds Wetenschappelijk Onderzoek) of Vlaanderen for support. B.P. is a postdoctoral fellow of FWO (Fonds Wetenschappelijk Onderzoek).

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