Loading Capacity versus Enzyme Activity in Anisotropic and Spherical

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The loading capacity versus the enzyme activity in new anisotropic and spherical vaterite microparticles Senem Donatan, Alexey M. Yashchenok, Nazimuddin Khan, Bogdan Parakhonskiy, Melissa Cocquyt, Bat-El Shani Pinchasik, Dmitry Khalenkow, Helmuth Möhwald, Manfred Konrad, and Andre G Skirtach ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b03492 • Publication Date (Web): 11 May 2016 Downloaded from http://pubs.acs.org on May 18, 2016

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ACS Applied Materials & Interfaces

The Loading Capacity Versus the Enzyme Activity in New Anisotropic and Spherical Vaterite Microparticles Senem Donatan1,‡, Alexey Yashchenok1,4,‡ *, Nazimuddin Khan2,‡, Bogdan Parakhonskiy3,4,5‡,*, Melissa Cocquyt5, Bat-El Pinchasik1, Dmitry Khalenkow5, Helmuth Möhwald1, Manfred Konrad2, Andre Skirtach1,5 1

Department of Interfaces, Max Planck Institute of Colloids and Interfaces, Golm/Potsdam, D-14476, Germany,

2

Enzyme Biochemistry Group, Max Planck Institute for Biophysical Chemistry, Göttingen, D-37077, Germany, 3

4

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 5

Department of Molecular Biotechnology, NB-Photonics Group, Ghent University, Ghent 9000, Belgium

KEYWORDS vaterite, enzyme, calcium carbonate, polyelectrolyte, enzyme-catalyzed reaction

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 1 ACS Paragon Plus Environment


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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 harbouring 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 to the loading capacity and geometry. Our results reveal that the particles' shape influenced on morphology of particles and as result affects the activity of the encapsulated enzyme, 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.

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INTRODUCTION A promising strategy to enhance the cellular targeting efficacy in drug delivery systems is mimicking the biological behavior1, which is often related to shape-specific, anisotropic and non-covalent interactions between biological molecules2. In addition, the delivery of biomolecules can be enhanced by choosing a delivery vehicle of a desired geometry. Therefore, development of non-spherical carriers with elongated, filamentous morphologies3, or anisotropic delivery systems4–8, is regarded as an essential advance due, in part, to more effective cellular targeting9,10 and uptake11. Alteration of biological responses was reported for particles with similar compositions, but with different geometries12–14 entering the bio-distribution pathways15–17. 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 favourable hydrodynamic properties. The shape has also been identified to play a major role in the uptake of drug carriers into cells20. Capsules with cubical21,22 , semispherical22, 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 methods24–28, while fabrication of anisotropic calcium carbonate particles through controlling the ratio of salts also has been reported23,29. Such different anisotropic shapes of drug delivery carriers were shown to facilitate cell uptake37,25, i.e. the uptake of capsules by cells depended on the aspect ratio of particles and capsules. 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 which are often designed for mimicking naturally occurring delivery systems. Polymeric capsules engineered through the highly 3 ACS Paragon Plus Environment


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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 applications30–32. The LbL technique is based on the consecutive adsorption of oppositely charged polyelectrolytes on a preformed charged template core which is chemically dissolved after the coating process. Lbl capsules have already been widely used for encapsulation of biological substances especially enzymes like urease33,34, luciferase35, glucose oxidase and peroxidase33,36. 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 templates37. In particular, CaCO3 particles represent a very important class of templates for LbL-engineered capsules in drug delivery applications due to their highly porous structure, biocompatibility38, degradability in mild condition39 and ease of fabrication40. The shape of these CaCO3 templates is mainly spherical, and their crystal structure usually is vaterite. Among the three polymorphs (calcite, aragonite, or vaterite) of CaCO3, vaterite is thermodynamically the least stable one41. However, for drug delivery applications, it is an attractive candidate since vaterite particles are not toxic38 and highly porous capable of immobilizing many small39,42,43 and large molecules44–47 into their interior structure48. 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 additives49–52. If additives such as dipeptides52, polypeptides49, peptide-type block copolymers, or 4 ACS Paragon Plus Environment

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inorganic materials, which bind to peptides51, 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 of by adding polyethylene glycol together with changing the salt ratio: New shapes of particles, which have not been reported to date, resulted from this approach. A detailed scanning electron microscopy (SEM) study of particle surfaces and confocal laser scanning microscopy (CLSM) studies of loaded molecules have been conducted. Promising applications of such porous particles were recently identified in enzyme-catalyzed reactions56–58. 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, which has not yet been studied in depth.

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 dehydrate (CaCl2 .2H2O), sodium carbonate (Na2CO3), adenosine 5′-triphosphate (ATP), guanosine 5′-monophosphate (GMP), pyruvate kinase and lactate dehydrogenase were purchased from Sigma-Aldrich, Germany. Nicotinamide adenine 5 ACS Paragon Plus Environment


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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 min, 60 min, 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 10 mg of vaterite particles immersed in water during 24 h. 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 technique60,61. For this, pre-synthesized CaCO3 particles (5 mg) were incubated with 2 ml of polyelectrolyte solution during 15 minutes. 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 6 ACS Paragon Plus Environment

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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 0.1M TRITC-dext solution was added to 10 mg of presynthesized particles. After 30 min 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 p0,8

0,6± 0,3

6.8

4

32

Small

0,6 ± 0,1

22

6,3

4 ±0,9

7

28

42

Small

0,8±0,2x

30

3,5

3 ±0,2

23

69

33

elliptical

0,8±0,2x

3,6 ± 0.7

15-22

1,0

2,8 ±2,3

28

78

49

4 ± 0,5

18

>1,3

4,8 ±0,9

31

148

37

spherical

1,4± 0,3 Large spherical Star-like


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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%). Since 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 weight percent. 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 enzyme-loaded 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: ~ × −

(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. The activity of enzymes encapsulated in 1 mg of calcite (rhomboidal) particles is the lowest, consistent with the lowest loading 13 ACS Paragon Plus Environment


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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 polystyrene sulfonate (PSS), and a subsequent layer of polyallylamine hydrochloride (PAH), and (iii) with 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 non-coated 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 twice higher zeta 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 on 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 14 ACS Paragon Plus Environment

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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 release70; this can be achieved by varying the density of charges71, or by the number of applied polyelectrolyte layers72,73. The catalytic activity of enzyme-loaded 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 the 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 product molecules (here: ADP, GDP). As such, covering particles by polyelectrolyte multilayers is an effective method to control the enzymatic activity.

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 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 15 ACS Paragon Plus Environment


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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 influence 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 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, while 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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FIGURES

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 (gi) medium. The scale bars on images correspond to 2 µm.



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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 b,d images are 2 µm, and in c image it is 10 µm.


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

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) 19 ACS Paragon Plus Environment


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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 oC 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 1ml 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 1ml 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.


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AUTHOR INFORMATION Corresponding Author *Corresponding authors: Bogdan Parakhonskiy: [email protected]; Department of Molecular Biotechnology, NB-Photonics Group, Ghent University, Ghent 9000, Belgium Alexey Yashchenok: [email protected] Max Planck Institute of Colloids and Interfaces, Golm, Germany; Remote Controlled Theranostic Systems Lab, Institute of Nanostructres and Biosystems, Saratov State University, 410012 Saratov, Russia

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Funding Sources Government of the Russian Federation (grant №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 №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 Vlaanderen for support. We also thank the Marie-Curie IRSES project “DINaMIT” and ERA.Net Rus project “IntelBioComp”. ACKNOWLEDGMENT We would like to thank Rona Pitschke for SEM images, Ingrid Zenke for X-ray diffraction measurements. S. D. thanks TUBITAK. A.M.Y thanks the Alexander von Humboldt-Stiftung. A.M.Y 21 ACS Paragon Plus Environment


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and B.V.P acknowledge the Government of the Russian Federation (grant №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 №15-29-01172 ofi_m and postdoctoral program of FWO (Fonds Wetenschappelijk Onderzoek). N.K. has been supported by a DAAD PhD 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). Supporting Information. The XRD data of spherical, ellipsoidal and rhomboidal CaCO3 particless is available in Supporting Information.

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and without Layer-by-Layer Thin Coatings. Polymers (Basel). 2014, 6 (8), 2157–2165.

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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. 107x75mm (300 x 300 DPI)

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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 b,d images are 2 µm, and in c image is 10 µm. 79x47mm (300 x 300 DPI)



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Figure 3. SEM images of s rhomboidal - and star-like shaped calcium carbonate particles. The scale bars correspond to 2 µm. 39x19mm (300 x 300 DPI)


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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 oC 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 1ml 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 1ml 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 (υ) 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. 69x27mm (300 x 300 DPI)



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Graphical abstract 43x15mm (300 x 300 DPI)


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Loading Capacity versus Enzyme Activity in Anisotropic and Spherical

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