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Materials for biology and medicine

Journal ofMaterials Chemistry Bwww.rsc.org/MaterialsB Volume 1 | Number 9 | 7 March 2013 | Pages 1201–1372

PAPERAlexey Yashchenok et al.Polyelectrolyte multilayer microcapsules templated on spherical, elliptical and square calcium carbonate particles

www.rsc.org/MaterialsBRegistered Charity Number 207890

Showcasing research from the Department of

Chemistry and Nano Science, Ewha Womans

University, Seoul, South Korea.

Title: Photoluminescent nano graphitic/nitrogen-doped graphitic

hollow shells as a potential candidate for biological applications

Water-dispersible nanosized graphitic/N-doped graphitic hollow

spheres were successfully synthesized using a soft chemical route.

Cellular uptake of the graphitic/N-doped graphitic hollow spheres

was evaluated in human HeLa cells, demonstrating its main

localization in the cytoplasm, and blue and green fl uorescence

signals were observed with low toxicity in the cells.

As featured in:

See J.-E. Park et al.,

J. Mater. Chem. B, 2013, 1, 1229.

Journal ofMaterials Chemistry B

PAPER

aDepartment of Interfaces, Max-Planck

Golm/Potsdam, D14476, Germany. E-mail:bItaly BIOtech, University of Trento, MattarecShubnikov Institute of Crystallography, RusdDepartment of Molecular Biotechnology, GheNB-Photonics, Ghent University, Ghent 900

† Electronic supplementary informa10.1039/c2tb00416j

Cite this: J. Mater. Chem. B, 2013, 1,1223

Received 15th November 2012Accepted 27th December 2012

DOI: 10.1039/c2tb00416j

www.rsc.org/MaterialsB

This journal is ª The Royal Society of

Polyelectrolyte multilayer microcapsules templated onspherical, elliptical and square calcium carbonateparticles†

Alexey Yashchenok,*a Bogdan Parakhonskiy,bc Senem Donatan,a Dorothee Kohler,a

Andre Skirtachade and Helmuth Mohwalda

Recent studies have revealed that a variety of shaped particles can interact with cells in a different way.

Elongated particles can be effectively and quickly internalized intercellularly compared with other

configurations. Herein we present the potential of fabrication of anisotropic polyelectrolyte multilayer

capsules formed by coating spherical, ellipsoid-like and square calcium carbonate (CaCO3) particles. By

varying the intermixing speed, time, pH value and ratio of initial ingredients during precipitation such

CaCO3 templates are produced. Particles loaded with FITC–dextran and coated with polyelectrolytes

could maintain the templated shape after core removal. Quantitative data are derived from analysis of

confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) measurements.

Introduction

Designing novel drug delivery carriers, including those with aspecic geometry, gained increasing attention during the pastdecade. Indeed, shape, surface chemistry, charge and size wereshown to have a major impact on particle fate in vivo.1 Bio-distribution and interaction pathways aer particle adminis-tration are not clearly understood to date. Geng et al. investi-gated the inuence of particle shape on blood circulation,showing ten fold prolonged blood circulation for lomicellescompared to spherical particles.2 Furthermore, increasedmuco-adhesion in the gastrointestinal tract due to the presence ofnanobers on glass beads was reported, with increased van derWaals forces and thereby increased surface area.3 Championet al. studied the macrophage response to various particleshapes produced by a stretching based technique.4 The targetgeometry at the point of the rst cell contact thereby inuencesthe efficiency of uptake, and determines the uptake kinetics, asinternalization of worms was found to be 20 times slowercompared to spheres of the same volume.5 The particle shape istherefore a main parameter inuencing the particle uptake,while the particle size has a major impact on the success of

Institute of Colloids and Interfaces,

alexey.yashchenok@mpikg.mpg.de

llo, Italy

sian Academy of Science, Moscow, Russia

ent University, Ghent 9000, Belgium

0, Belgium

tion (ESI) available. See DOI:

Chemistry 2013

phagocytosis, as complete phagocytosis is only observed forparticle volumes smaller than the macrophage volume.6

Polyelectrolyte microcapsules have established a reputationas a carrier for drug delivery in one row with other types such asnano- and microparticles, liposomes and red blood cells.7,8 Thelayer-by-layer (LbL) technique allows production of multilayercapsules of various shell compositions at the nanometer scale,and that increases functionality and external response.9–12

Multicompartmentalization of capsules has recently become anattractive route to enhance the modularity of polyelectrolytevesicles, which could be used for carrying various cargoessimultaneously and for conduction of biomimetic reactions.13–15

In this context researchers have elaborated on anisotropic andhierarchical nano- and microcapsules with properties thatcould not be achieved using conventional approaches.16–20 Ageneral procedure of capsule fabrication is based on adsorptiononto an organic or inorganic core with oppositely chargedpolyelectrolytes and subsequent dissolution of the templatedparticle.21,22 The capsule conguration depends on the size andshape of the initial core, thus templates with a dened structureenable fabrication of a variety of capsule congurations.23–26 Forexample, Shchepelina et al., had studied the morphology,mechanical properties, and permeability of hydrogen-bondedlayer-by-layer (LbL) microcapsule shells assembled on cubiccadmium carbonate (CdCO3) cores in comparison with thetraditional shells assembled on spherical silica microcores.27

The patterned template-assisted assembly of the cubic micro-particles driven by the competing capillary, coulombic, and vander Waals forces in comparison with the traditional sphericalcolloidal microparticles had been studied by Lisunova et al.28

Among the numerous particles of polystyrene (PS), methylformaldehyde (MF), silica (SiO2), and others, calcium carbonate

J. Mater. Chem. B, 2013, 1, 1223–1228 | 1223

Journal of Materials Chemistry B Paper

(CaCO3) is particularly attractive because of its native biomineralcomposition which is abundant in nature.29,30 A variety ofcapsules was fabricated based on CaCO3 templates due to theirbiocompatibility, high loading capacity and easiness in prepa-ration. These features are essential not only for industrial appli-cations, for example as ller in paints or papers,31 but also for awide range of other applications, such as drug delivery carriers,32

biomolecule microreactors,13,15 and periodic nano- and micro-structures.33,34 Numerous calcium carbonate particles withvarious shapes were introduced by using additives.18,35,36 In thisway growthof carbonate particles canbe controlledby additionofpolymers, for instance, additionof poly-acrylic acid (PAA) inhibitsthe synthesis of CaCO3 microparticles.35 Three types of poly-morphs, calcite, vaterite and aragonite, have been controllablyproduced in the presence of poly(sodium 4-styrene sulfonate-co-N-isopropylacrylamide) (PSS-co-PNIPAAM).36Variationof calciumand polyelectrolyte concentrations enables a systematic controlover the size and morphology of particles among which are:pseudo-dodecahedra, pseudo-octahedra, multilayered spheres,and hollow spheres.37 In general, calcium carbonate exists in theform of several polymorphs: calcite, vaterite, and aragonite.38

They have typical morphologies: calcite – rhombohedral, arago-nite – needle, and vaterite – spheres. The rst two morphologiesare most oen found in biological organisms, which use suchmaterials for building-up of their shells. Moreover, those phasesof calcium carbonate are thermodynamically most stable incomparison to vaterite, which is ametastable polymorph and notcommonly formed by organisms.39 On the other hand, vaterite isan attractive candidate because of its high surface area andabundance of pores. The latter makes vaterite appropriate forentrapping biological molecules and drugs in the porous struc-ture for delivery and release of cargo by means of particlesthemselves or capsules within cells, tissue or in the body.32,40

In this work, we present modied approaches to producevarious polymorphs of calcium carbonate particles (spherical,elliptical, and square). Ellipsoid-like CaCO3 particles wereproduced by xing of the salt ratio and pH in an ethylene glycolwater solution.We obtained rhombohedral calciumpolymorphsin two ways: the rst by long-timemixing of initial salt solutions,and the second by low-speed stirring. Such calcium carbonatetemplates were used for capsule fabrication via the layer-by-layer(LbL) assembly with frequently used polyelectrolytes, whileloading was conducted withmodel FITC–dextranmolecules. Wedemonstrated that the initial texture of these types of anisotropiccores is affected to arrive at a nal morphology of the hollowmicrocapsules using a similar release of the solid CaCO3 core.This study represents the rst step in designing biocompatibleanisotropic calcium carbonate templates and synthetic capsuleswithout any additives. It should be stressed that development ofsuch carriers is expected to provide a broad range of applicationsincluding pulmonary drug delivery.

Experimental sectionMaterials

Poly(allylamine hydrochloride) (PAH, Mw 70 kDa), poly(sodium4-styrenesulfonate) sodium salt (PSS, Mw 70 kDa), uorescein

1224 | J. Mater. Chem. B, 2013, 1, 1223–1228

isothiocyanate–dextran (FITC–dex, Mw 70 kDa), ethyl-enediaminetetraacetic acid (EDTA), calcium chloride dihydrate(CaCl2$2H2O), and sodium carbonate (Na2CO3) were purchasedfrom Sigma-Aldrich, Germany. Ethylene glycol solution (99%)was purchased from Alfa Aesar. In all procedures MilliQ waterwas used with a resistivity of >18 MU.

Calcium carbonate particle synthesis

Calcium carbonatemicroparticles of variousmorphologies werefabricated using a modied previously reported protocol.41 Forall particles we used Na2CO3 and CaCl2$2H2O aqueous saltsolutions. Spheroid-like CaCO3 microparticles were producedas follows: 1 mL of Na2CO3 (0.33 M) was injected with agitationinto a glass vessel and then an equal volume of CaCl2 (0.33 M)was injected and stirred at 600 rpm for 1 min. The mixedsolution turned opaque almost instantly. Then, the slurry wasthoroughly washed with deionized water and dried in aconventional oven at 70 �C for 1 hour.

For ellipsoid-like CaCO3 particles we used the procedurewith a water–ethylene glycol combination:42 1 mL of Na2CO3

(1 M) was injected into a glass vessel containing ethylene glycol(EG) (8 mL EG) and then 1 mL of CaCl2 (0.1 M) in 8 mL EG wasadded and stirred at 600 rpm for 10, 30, and 90 min. Beforeparticle preparation the pH value of the mixing solutions ofNa2CO3 and CaCl2 was adjusted in the range 9–9.5. The solutionturned opaque aer several minutes when it was mixed. Theresulting solution was then thoroughly washed with water andalcohol several times in order to remove residual ethyleneglycol. The same drying procedure was performed as in the caseof spherical CaCO3 particles.

Particles with cubic-like morphology were prepared asfollows: in the glass ask containing 1 mL of Na2CO3 (1 M) anequal volume of CaCl2 (1 M) was injected (with agitation) andthe mixture was further stirred at 600 rpm for �3 hours. Thenthe nal mixture was washed with water several times in orderto remove the un-reacted components. Another way to preparesquare particle morphology was used. In the glass ask con-taining 1 mL of Na2CO3 (0.01 M) a solution containing 1 mL ofPAH–FITC (2 mg mL�1) was added under agitation. Then anequal volume of CaCl2 (0.01 M) was injected, and the mixturewas stirred at a low rate (200 rpm) for 10 seconds. Then the nalmixture was placed on lter paper and washed with deionizedwater to remove the unreacted components.

Capsule fabrication

The shape of microcapsules is determined by the shape of theoriginal CaCO3 templates. The synthetic polyelectrolytes PAHand PSS were subsequently adsorbed onto the particle surfaceusing the layer-by-layer (LbL) deposition technique from solu-tions containing 2 mg mL�1 of molecules dissolved in 0.15 MNaCl. PAH was rst adsorbed onto the spherical and ellipsoidparticles, while for square cores PSS was used as the initial layer.For visualization of the capsules dye labeled molecules (FITC–dextran) were incorporated into the interior of the particles byadsorbing those molecules in the pores. To obtain hollowcapsules the CaCO3 core was decomposed by EDTA. EDTA

This journal is ª The Royal Society of Chemistry 2013

Paper Journal of Materials Chemistry B

solution with a concentration of 0.2 M at pH 7 was added andthe particles were collected by centrifugation. Further EDTA wasremoved by washing with deionized water several times.

Characterization

Optical microscopy images were recorded with a Leica TCS SPconfocal scanning microscope (Leica, Germany) in invertedmicroscope mode equipped with a 100�/1.4–0.7-oil immersionobjective.

Scanning electron microscopy (SEM) images were recordedby means of a Philips XL30 electron microscope at an acceler-ating voltage of 3 kV.

Results and discussion

We fabricated microcapsules using calcium carbonate micro-particles as sacricial templates. Calcium carbonate micropar-ticles have become the material of choice for production ofbiologically compatible particles and capsules. The particles arefabricated by stirring salts which form a precipitate. Stirring istypically conducted at a high speed, and that leads to formationof spherical particles.

Fig. 1 shows SEM images of CaCO3 polymorphs: vaterite(spheroids and ellipsoids), and calcite. Vaterite has porousmorphology in both spherical and ellipsoid-like structures(Fig. 1a and b). The average size of the spheroid-like particles isin the range of 4–6 micrometers, which is consistent withprevious studies.43 Ellipsoid-like particles are smaller thanspherical ones, and their average size (axis ratio) depends on theratio of calcium chloride to sodium carbonate and the pH valueof the mixing solutions. In our experiments, we used a pH in therange of 9–10 for both calcium chloride and sodium carbonatesalts. Another parameter that can inuence the shape of CaCO3

microparticles is the ratio of salt concentrations (molarities ofinitial solutions of Na2CO3 and CaCl2). For ellipsoid-like struc-tures the following salt ratios were used: 2.5, 5, and 10. We havefound that at the short stirring period (about 10 min) the axisratio of the CaCO3 particles is independent of the initial saltratio; with increasing agitation time we obtained an increase inthe size of ellipsoid-like particles. During 30 minutes of agita-tion of calcium chloride and sodium carbonate salt solutionswith ratios: 2.5, 5, and 10, we observed the following aspectratios: 1.4, 1.6, and 2.0, respectively. Fig. 1b shows that mostcalcium carbonate particles are ellipsoid-like. Meanwhile,

Fig. 1 SEM images of CaCO3 microparticles: (a) spherical particles (vaterite),fabricated according to ref. 41; (b) ellipsoid-like structures (vaterite), stirring for 90min; and (c) rhombohedral microcrystals (calcite), synthesized for 2 hours. Thescale bars in all images correspond to 2 micrometers.

This journal is ª The Royal Society of Chemistry 2013

irregular stars are also observed together with oval particles. Inorder to determine the number of ellipsoid-like and owerparticles in the same volume we analyzed more than 100particles from the SEM pictures. We found that about 10% ofall particles are irregular stars.

The pH value of the mixing salt solutions plays the mainrole in formation of ellipsoid-like particles. In this pH rangethe nucleation rate is high, and can lead to uncontrolledCaCO3 morphology. The solution supersaturation increasesbecause of the hydrocarbonate–carbonate buffer equilibrium,coupled with an increasing nucleation rate of calciumcarbonate.35 Another factor that can inuence the morphologyat this pH range is the secondary nucleation due to super-saturation towards the shape modication.35 Fig. 1c presentsrhombohedral CaCO3 microparticles. As can be seen from thisgure calcite morphology reveals low density or the absence ofpores compared with vaterite calcium carbonate particles.Such polymorphs are more stable than those of the vateritestructure. Moreover, calcite particles are present together withvaterite particles when those particles are freshly prepared andthen transformed to calcite in time, as this is thermodynam-ically favoured. Fig. 2 shows the change of the calcite micro-crystal fraction as a function of time. It can be seen from thisgure that at a short stirring time (�60 min) the production ofcalcite is below 20%, but it reaches almost 100% aer twohours. Another way to produce calcite CaCO3 particles isprecipitation upon rotation at a low speed (for example,200 rpm). For such a procedure we used the same salt solu-tions but with low concentration, as in the normal experimentthese salts are admixed together with additives. Aer such aprocedure smaller calcium carbonate particles are assembledinto a larger calcium carbonate particle also entrapping insideany added polymers or proteins. In our approach a lesserspeed of intermixing is used instead of vigorous stirring.Under such a procedure, calcium carbonate particles aregetting assembled into non-spherical, square in this case,particles.

As it was discussed above for various biological applica-tions (for example: transport and dynamics of interactions

Fig. 2 Calcite microcrystal amount as a function of stirring time in watersolution. The quantity of particles was estimated by image processing of SEMimages (at least 100 microparticles were counted per sample).

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Journal of Materials Chemistry B Paper

with the cells, articial carriers for drug delivery) it is desir-able to produce anisotropic (non-spherical) particles andcapsules. In this regard, differently shaped calcium carbonateparticles were covered with oppositely charged polyelectrolytesvia the LbL method. Confocal microscope images of threetypes of CaCO3 particles are shown in Fig. 3. We used uo-rescently labeled dextran molecules as a model system forencapsulating molecules within the porous particles. One canclearly see that all particles are loaded with FITC–dextranmolecules. However, in the case of square particles the uo-rescence intensity is lower compared with spheres and ellip-soids. This indicates a too small loading capacity for suchtypes of calcium carbonate particles compared to that forspherical and elliptical cores. The porous structure of vateriteCaCO3 particles (spheres and ellipsoid-like; SEM images)allows easy entrapment of the FITC–dextran molecules in theirpores and achievement of a relatively high capacity. Squarecalcium carbonate particles are monocrystals without porositydifferent from vaterite polycrystals. Hence calcite could notprovide sufficient loading of the FITC–dextran molecules intheir volume. Only a small amount of dextran molecules canbe adsorbed on the surface of square particles owing tonegligible roughness of the calcite structure. In order to ach-ieve capsules we dissolved the CaCO3 substrates by immersingthose particles into EDTA solution. The scanning electronmicroscopic images (Fig. 4) show spherical, ellipsoid, andsquare polyelectrolyte capsules, which were templated on theappropriate CaCO3 microparticles. One can see that poly-electrolyte lms adsorbed onto the surface of CaCO3 repro-duce the core shape and maintain the prole of the core aerdecomposition of the substrates. The surface morphology of

Fig. 3 Confocal laser scanning fluorescence images of spherical (a), ellipsoid-like(b) and square (c) CaCO3 microparticles. The microparticles were embedded withFITC–dextran molecules by co-precipitation and subsequently covered by severaloppositely charged polyelectrolyte layers through the LbL assembly. The scalebars in all images correspond to 5 micrometers.

Fig. 4 SEM images of hollow anisotropic microparticles comprised four bilayersof polyelectrolytes PAH–PSS templated on (a) spherical; (b) ellipsoid-like; and (c)square CaCO3 microparticles. The scale bars in all images correspond to 4micrometers.

1226 | J. Mater. Chem. B, 2013, 1, 1223–1228

the spherical microcapsules reveals a rough structure withfolds and holes (Fig. 4a). This indicates that the polyelectrolytelm repeats the surface morphology of the CaCO3 micropar-ticles, in accordance with observations reported by othergroups.43 The ellipsoid-like microcapsule morphology showsalso folds. However, the surface of the ellipsoid microcapsulesis smoother compared to spheres, which indicates formationof a more compact polyelectrolyte lm. The surfacemorphology of square microcapsules is shown in Fig. 4c. Thistype of capsules also has holes, however the polyelectrolyteshell appears thinner than spherical and ellipsoidal micro-capsules. This can be attributed to the surface texture of therombohedral CaCO3 microparticles (Fig. 1c). As we have dis-cussed above square microparticles have not enough poroussurface, which could allow them to effectively adsorb poly-electrolytes. The latter could also alter adsorption of the rstpolyelectrolyte layer. As a sequence this fact might be criticalfor forming a stable polyelectrolyte complex.

In order to verify how the microcapsule shell behaves in theabsence of the solid core we made a quantitative analysis ofthe size distribution before and aer template dissolution.Counting was performed by using processing of confocaltransmission images with the ImageJ program.44 For analysiswe have counted at least 150 particle capsules. We obtained agood correlation between the size of the initial anisotropicCaCO3 particles and the size of the microcapsules when thecore was removed. The size of the spherical particles wasestimated to be 3.67 � 0.07 mm, whereas the size of the samecapsules aer core removal is 3.63 � 0.08 mm. The sametendency was observed for the square templates: sizes of thecore and the capsule are 4.00 � 0.10 mm and 4.10 � 0.10 mmrespectively. In the case of the ellipsoidal size of the micro-capsules the long axis was found to be 1.70 � 0.05 mm,while the length of the microcapsules is decreased to 1.48 �0.04 mm.

One would expect that these types of porous anisotropicCaCO3 particles will potentially be useful as templates toproduce polyelectrolyte microcapsules45–48 and as carriers withtunable release of encapsulated macromolecules49–53 and ther-apeutics.54 We also believe these systems can be applied forcarrying various substances simultaneously and for conductionof biomimetic reactions.13,14 The approach proposed herecould also be benecial to development of label-free Ramanprobes for detection of biomarkers55 and tracing intercellularmolecules.56

Conclusions

We presented the results of polyelectrolyte multilayer capsulefabrication with a variety of morphologies templated fromcalcium carbonate particles. Spherical, elliptical and squareCaCO3 particles can be synthesized through changing the stir-ring speed, time, pH value and salt ratio. Stirring for three hoursyields amounts of calcite crystals of almost hundred percent.Polyelectrolyte capsules can be fabricated over such anisotropiccalcium carbonate templates simultaneously loaded with FITC–dextran molecules in their pores. Polyelectrolyte shells of the

This journal is ª The Royal Society of Chemistry 2013

Paper Journal of Materials Chemistry B

hollow microcapsules can be reproduced with the surfacemorphology of anisotropic CaCO3 microparticles aer decom-position of those particles in EDTA solution. The surfacemorphology of the microcapsules reproduces the initial surfaceof the templated particles, which is conrmed by SEM data. Thecapsule shape can be retained similar to that of the initialtemplates presenting opportunities to construct anisotropicexible structures.

Acknowledgements

We thank Rona Pitschke for SEM images. A. Yashchenok thanksthe Alexander von Humboldt-Stiung for a scholarship.B. Parakhonskiy thanks the Marie-Curie foundation for thefellowship.

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