Feature Article
Multicompartmental Micro- and Nanocapsules: Hierarchy and Applications in Biosciences Mihaela Delcea,* Alexey Yashchenok, Kristina Videnova, Oliver Kreft, Helmuth Mo ¨hwald, Andre G. Skirtach*
Multicompartmentalized micro- and nanocapsules allow simultaneous delivery of several vectors or biomolecules; they are the next generation of carriers with increased complexity. Here we overview multicompartment micro- and nanocapsules and present a road-map for future developments in the field. Four basic building block structures are demonstrated, three isotropic: concentric, pericentric, and innercentric, and one anisotropic: acentric. As an elaborate implementation of multicompartmentalization, an enzyme-catalyzed reaction inside the same capsule carrying both an enzyme and a substrate is shown. Applications of multicompartmentalized microcapsules for simultaneous multiple drug delivery in bio-medicine are discussed.
Introduction Nanotechnology is playing an increasingly important role in drug delivery and bio-medicine.[1] It allows construction of sophisticated micro- and nanocapsules with increasingly complicated structures and functionalities. As technology approaches the nanometer scale, such new features as multifunctionality, remote addressability, mechanical stability, and multicompartmentalization emerge. The latter is envisioned to be the next area of development, first and foremost due to possibilities of simultaneous delivery of various drugs, medicines, or other chemicals. In fact, its application area is much broader because of possibilities of conducting biochemical, enzymatic, and proteomic reactions in confined volumes.[2] Some M. Delcea, A. Yashchenok, K. Videnova, O. Kreft, H. Mo ¨hwald, A. G. Skirtach Max-Planck Institute of Colloids and Interfaces, Research Park Golm, Potsdam 14424, Germany Fax: 0049331567 9202; E-mail:
[email protected];
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approaches to multicompartmentalization can be demonstrated using various encapsulation technologies, for example, polymeric micelles,[3] polymeric microspheres,[4] two-compartment vesicles.[5] In general, polymeric microcapsules were shown promising for biomedicine.[6,7] Polyelectrolyte multilayer capsules, a particular type of polymeric capsule, are ideally suited for analysis of multicompartmentalization because of the versatility of their design and composition. Indeed, polyelectrolyte multilayer capsules have undergone rapid development in the past decade due to possibilities of inclusion of a high amount of molecules, tailoring their composition, structural, physico-chemical, and mechanical properties at the nanometer scale.[8–17] They are fabricated using the layer-by-layer technique[18–24] by coating colloidal templates followed by core dissolution. Polyelectrolyte microcapsules have evolved from just experimental exploration to structures capable of delivering different types of molecules, therapeutic agents for delivery, and for diagnostic assays within their interior or on their surface.[25–27] For instance, polyelectrolyte multilayer microcapsules doped with gold nanoparticles in
DOI: 10.1002/mabi.200900359
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Mihaela Delcea received her M.Sc. in Microbiology and Biotechnology from Bucharest University, Romania in 2006 and worked as a junior researcher at Institute of Biology, Romanian Academy of Sciences. She completed her Ph.D. in 2009 at the CICbiomaGUNE Institute, San Sebastian, Spain. Her Ph.D. work was focused on bioengineering biomimetic membranes by combining proteins, polyelectrolytes, and lipids. Currently she is a postdoctoral researcher in the group of Dr. Skirtach at the Max Planck Institute of Colloids and Interfaces, Potsdam, Germany. Her work is focused on polymeric microcapsules and films, nanoparticles and nanotechnology, drug delivery and release, intracellular functions of molecules, and biomaterials for intracellular delivery and cell cultures. Alexey Yashchenok completed his Ph.D. in solid state electronic, radio-electronic components, micro- and nanoelectronics, and devices to investigate quantum effects in 2007 at Saratov State University, Russia with Professor Klimov. At present he is an Associate Professor at the Department of nano- and biomedical technology, Saratov State University. Recently, he visited the group of Dr. Skirtach at the Max Planck Institute of Colloids and Interfaces, Potsdam, Germany. His scientific interests are the design of nanocomposite structures including coatings and microcapsules by layer-by-layer methods, and investigation of their properties against their chemical composition and external environment. Kristina Videnova received her B.Sc. in chemistry with Marketing from Reutlingen University of Applied Sciences, Germany in 2007. She then obtained a M.Sc. in biochemistry from the University in Dusseldorf, Germany in 2009, completing the majority of the courses at the Research Centre Ju ¨lich, Germany. During the studies she also stayed at the Max Planck Institute of Colloids and Interfaces in Potsdam, Germany in the group of Dr. Skirtach, working on encapsulation of enzymes into microcapsules for pharmaceutical applications. She is currently working at the Veterinary Medicines Unit of the European Medicines Agency. Oliver Kreft completed his Ph.D. in biochemistry in 2003 at Potsdam University, Germany with Prof. L. Willmitzer. He stayed at the Max Planck Institute of Colloids and Interfaces (Potsdam, Germany) from 2004 to 2008 in the groups of Professor Sukhorukov and Professor Mo ¨hwald. In this period he focussed on the development of multifunctional microcapsules for biochemical and pharmaceutical applications, capsule-based sensor systems, and multicompartment capsules. Since 2008 he has been working as project leader for R&D in surface chemistry at Xella. Helmuth Mo ¨hwald received his Diploma in physics in 1971 at the University of Go ¨ttingen, Germany, and his Ph.D. in physics in 1974 at the Max Planck Institute of Biophysical Chemistry, Go ¨ttingen. In 1978, he received his habilitation at the University of Ulm, Germany. He was a C3 Professor at the Technical University of Mu ¨nchen, Germany, and held a Chair of Physical Chemistry (C4 professor) at the University of Mainz, Germany. Since 1993, he has been a director and scientific member at Max Planck Institute of Colloids and Interfaces, Potsdam, Germany. Among his recent awards were the Overbeek Medal of the European Colloid and Interface Society (2007), an Honorary Doctorate of the University Montpellier, France (2008), and the Wolfgang–Ostwald Medal of the German Kolloid-Gesellschaft (2009). His main research interests include biomimetic systems, chemistry and physics in confined spaces, dynamics at interfaces, and supramolecular interactions. Andre Skirtach received a M.Sc. degree in physics from Moscow State University, Russia in 1993 and a Ph.D. in chemistry from McGill University, Montre´al, Que´bec, Canada in 1997. Subsequently, he joined the National Research Council of Canada (Conseil national de recherches du Canada) in Ottawa, Ontario as an associate research officer. In 2000, Dr. Skirtach and colleagues transferred to industry technology developed at NRC-CNRC and founded a prominent start-up company— Trillium Phot., Inc. Subsequently, he moved to the Max Planck Institute of Colloids and Interfaces, Golm, Germany, later becoming a group leader in the Department of Interfaces (Professor Mo ¨hwald). His scientific research interests include nanotechnology and its application in biology, nanoparticles and their interaction with external fields (electromagnetic, laser, magnetic, ultrasound), polymers and polymeric capsules and planar interfaces, and self-assembly and its applications.
their walls were used for the intracellular delivery and controlled release of small peptides. Capsules loaded with peptides through thermal shrinking were introduced by electroporation into living cells, and were subsequently opened by an infrared laser pulse. The released peptides further bound to the major histocompatibility complex (MHC) class I proteins to accomplish their cell surface transport and antigen presentation.[28] In another experiment, De Kokker and De Geest used biodegradable dextran sulfate–poly(L-arginine) polyelectrolyte capsules for the delivery of antigen presenting cells (APCs) both by the MHC I and MHC II routes since they are readily taken up by
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dendritic cells in vitro and appear quite resistant to extracellular degradation.[29] Caruso et al. also used polyelectrolyte capsules to successfully target colorectal cancer.[30,31] In this case, capsules coated with huA33 monoclonal antibody were internalized by colorectal cells expressing the A33 antigen, a transmembrane glycoprotein that is expressed by 95% of all human colorectal tumor cells. As these and new applications are developed, new features such as multicompartmentalization are sought. It can be noted though that, besides introducing additional functionalities for delivery vehicles and augmenting a
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number of possibilities for studying reactions, multicompartmentalization can be indispensable in some biomedical applications where simultaneous delivery of several molecules is needed. However, to the best of our knowledge, there has been no systematic study of various strategies leading to multicompartmentalization, its hierarchy, building blocks, and diversity. In this work we overview multicompartment micro- and nanocapsules and outline future directions in the field by providing a road-map of trends and developments. The feasibility of four distinguished approaches to multicompartmentalization is demonstrated and their advantages and drawbacks are discussed. We also present the results of implementation of multicompartmentalization strategies wherein such an approach is indispensable: performing an enzyme-catalyzed reaction in the same multicompartment capsule. It can be noted that, although some enzymatic reactions have been performed before, our concept of incorporating both the enzyme and substrate in the same capsule is novel since in previously reported work[32,33] the substrate was not encapsulated but was freely floating in the solution with the microcapsules.
Experimental Part CaCO3 particles were prepared according to the previously described procedure.[32] Briefly, 1 M CaCl2 (0.615 mL) and 1 M Na2CO3 (0.615 mL) were rapidly mixed and thoroughly agitated on a magnetic stirrer for 20 s at room temperature. If the reaction time is reduced to several seconds, smaller CaCO3 particles are produced. After the agitation, the precipitate was separated from the supernatant by centrifugation and washed three times with Milli-Q water (resistivity higher than 18.2 MV cm). The resulting CaCO3 particles were co-precipitated with tetramethylrhodamineisothiocyanate (TRITC)-human serum albumin (HSA) and magnetite nanoparticles. This precursor was further coated with five bilayers of polyelectrolyte using the layer-by-layer (LbL) deposition technique[8–15] by alternating the adsorption of negative PSS (poly(sodium 4-styrenesulfonate); Mw ¼ 70 kDa) and positive PAH (poly(allylamine hydrochloride); Mw ¼ 70 kDa) (2 mg mL1 in 0.5 M NaCl). Further, the initially coated cores were subjected to the second co-precipitation step in the presence of Alexa Fluor 488. The AlexaHSA particles were collected by applying a magnetic field, while the non-magnetic co-products were washed out. By depositing five bilayers of PSS and PAH, concentric ball-in-ball particles were formed. The CaCO3 core was removed by complexation with ethylenediaminetetraacetic acid, EDTA (0.2 M, pH 7.5) as described previously[34,35] leading to the formation of shell-in-shell capsules. Scanning electron microscopy (SEM) images of shell-in-shell capsules were recorded with a Gemini 1550 (Zeiss, Oberkochen, Germany) at the acceleration voltage of 3.0 KeV. A drop of the capsule suspension was placed on a glass slide and allowed to dry at room temperature. To examine their interior, particles were slightly crushed.
Macromol. Biosci. 2010, 10, 465–474 ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
It can be noted that according to this strategy, particles can be aggregated first and then coated with additional shells, which leads to innercentric structures with multiple inner cores. Therefore, the first step for concentric and innercentric methods can be accomplished similarly. Aggregation can also be used to construct anisotropic structures, for example, for acentric multicompartment capsules. Acentric structures presented in this work have been achieved as a by-product of CaCO3 synthesis. For the pericentric approach, larger CaCO3 particles (diameter 5 mm) were prepared as described above and further resuspended for 2 h under shaking in tetramethylrhodamine isothiocyanate-dextran solution (TRITC-dextran, Mw ¼ 150 kDa) (1 mg mL1). The resulting CaCO3 particles loaded with TRITCdextran were subsequently coated with five PAH/PSS bilayers. Small polystyrene (PS) microparticles (Microparticles GmbH) with an average size of 1.08 0.04 mm were coated with three bilayers of PAH and PSS as described above. The second layer of PAH was labeled with FITC (fluorescein isothiocyanate). Adsorbing small PS particles (0.5 mL, 50 mg mL1) labeled with PAH-FITC onto large CaCO3 particles (0.1 mL, 50 mg mL1) with loaded TRITC-dextran led to pericentric constructs. Similar to the adsorption of PS particles, smaller CaCO3 particles were also adsorbed onto larger CaCO3 particles. Confocal laser scanning micrographs (CLSM) of capsules were recorded with a Leica system mounted to a Leica Aristoplan and equipped with a 100 oil immersion objective with a numerical aperture of 1.4. The enzymatic reaction in multicompartment microcapsules was conducted according to the following procedure. CaCO3 particles (5–6 mm in diameter) were incubated under agitation with peroxidase (POD, Sigma–Aldrich) (1 mg mL1 in 10 103 M TRIS buffer, pH 7.4) for 20 min at room temperature. After agitation, the mixture was washed three times in Milli-Q water. The resulting particles with loaded POD were then coated with five bilayers of PSS and PAH. CaCO3 dissolution was performed by treatment with EDTA as described above. The enzymatic activity of POD was estimated by adding 25 106 M H2O2 and 50 106 M Amplex Red in dimethyl sulfoxide (DMSO) to the capsule solution. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC, Sigma– Aldrich) liposomes were prepared by dissolving lipids in chloroform. After the evaporation of the organic solvent under a nitrogen stream and drying under vacuum overnight, a film of lipid molecules was formed. The obtained film was hydrated with HEPES buffer (10 103 M HEPES, 2 103 M CaCl2, 150 103 M NaCl, pH 7.4) that contained 0.75 103 M Amplex Red (Invitrogen) under vortexing in order to accelerate lipids to form vesicles in suspension. The obtained multilamellar liposomes were extruded several times through a polycarbonate membrane (100 nm diameter pore size) mounted in an extruder. Further, liposomes were dialyzed against HEPES buffer using a cellulose membrane with an exclusion pore size of 10 kDa. The liposomes were slightly positively charged and had sizes in the range of 152.3 4.6 nm. CaCO3 capsules with loaded POD were further incubated for 20 min with gentle shaking with DOPC liposomes that contained Amplex Red. The obtained suspension was then centrifuged and washed with Milli-Q water. Furthermore, the suspension was sonicated for 5 min at room temperature in a Sonorex Super bath of 240 W power (Bandelin electronic, GmbH & Co.KG). The reaction between Amplex Red and POD was monitored in situ by CLSM before and after sonication.
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trolyte capsules.[32] Such two-compartment capsules have exciting potential for biomedical applications due, in part, to possibilities of removing the separation between Our analysis of multicompartmentalization yields four compartments and thus following the course of biodifferent strategies: concentric, acentric, pericentric, and chemical reactions.[2] Remote opening of microcapsules innercentric (Figure 1). In this work, we show the feasibility of the implementation of all structures by demonstrating was demonstrated using magnetic fields,[37] ultrasound,[38] the first step for each approach. Each step can be further or IR lasers.[39–44] Also, it was performed using stimuli such extended by fabrication of additional compartments; as pH and polarity or ionic strength of the solvent.[45–48] An throughout this work we used CaCO3 templates due to interesting approach that can allow tracing diffusion of molecules is direction-specific release.[49] Although drug ease of fabrication and its general importance.[36] delivery can significantly benefit from such an approach, its The first approach consists of creating ‘concentric’ best application can be in studying reactions in multimulticompartment capsules. A single compartment serves compartments. The fabrication of such microcapsules can as the matrix for the formation of additional concentric be laborious, particularly if one needs to separate bycompartments. These structures are associated with products of the reaction.[32] Other results on multicom‘onion’-like multicompartment capsules (Figure 1a). Such systems present several advantages such as: i) unlimited partmentalization of polyelectrolyte capsules templated on number of sub-compartments, ii) efficient separation of the melamine formaldehyde and silica have been previously encapsulated materials, iii) tuning the permeability reported.[50] between various compartments, and iv) possibilities to We focus the discussion here on concentric and conduct reactions, for example, enzyme-catalyzed and pericentric strategies and briefly address the initial steps chain reactions. In addition, these systems can be used for of acentric and innercentric strategies. The calcium biomedical applications since they can be subjected to carbonate microparticles obtained by direct mixing of remote release of their content. The shell-in-shell microsoluble salts of Ca2þ and CO2 3 are homogeneously sized and capsules concept was first introduced by Kreft et al. as a tool highly porous. SEM images (Figure 2) illustrate the for spatially confined enzymatic reactions inside polyelecformation of concentrically built CaCO3 particle-in-particle multicompartments. The size of the particle-in-particle structure ranged from 8–10 mm and the inner core diameter was 4–5 mm (Figure 2b). Figure 2b represents a cross-section of a particle-in-particle structure and illustrates the radially symmetric character of the internal structure of the particles. Porosity is an important feature of CaCO3 particles. Because of its highly porous architecture, the effective surface area of the CaCO3 microparticles is greater than that of a compact particle. Porosity can be clearly observed in Figure 2a and b, the inner porosity is important for encapsulation of biomolecules (e.g., proteins, enzymes, and peptides), while the porosity at the outer surface is significant for immobilizing biomolecules, nanocapsules, nanoparticles, etc. When a particle-in-particle delivery vehicle is fabricated, one also produces single particles, so separation of these two types of delivery vehicles needs to be performed. Core dissolution of the CaCO3 particleFigure 1. Overview and the road-map for future directions of multicompartment microin-particle leads to the formation of shellcapsules. Four different approaches are identified in the schematics: a) concentric, b) pericentric, c) innercentric, and d) acentric. The structure in the middle incorporates all in-shell capsules (see Figure 3). It can be four approaches. The corresponding confocal microscope images of the first steps in seen that the outer compartment is filled each direction are also presented. Scale bars correspond to 2 mm. with Alexa Fluor 488-HAS (green color)
Results and Discussion
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Figure 4. SEM micrograph of dual-compartment microcapsules. The two compartments are separated by a polymeric shell surrounding the inner microcapsule.
Figure 2. SEM micrographs: a) single CaCO3 particle and b) crosssection of concentrically built CaCO3 particle-in-particle multicompartment.
while the inner part is filled with TRITC-HAS (red color). The inner and outer capsules can be clearly seen in Figure 4. These compartments are separated, which indicates that there is a polymeric shell surrounding the inner micro-
Figure 3. CLSM image of two-compartment microcapsules. The outer CaCO3 compartment is filled with Alexa Fluor 488-HSA (green color) and the inner one is loaded with TRITC-HSA (red color). Both compounds are separated by a polyelectrolyte shell. The scale bar is 5 mm. Macromol. Biosci. 2010, 10, 465–474 ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
capsule. Such two-compartment capsules can find potential applications in biomedicine. The separation between the compartments could be remotely eliminated, allowing mixing of their content.[2] The second approach is ‘pericentric’ multicompartment capsules (Figure 1b). Its first noticeable advantage is simplicity of fabrication and versatility of the structure. Indeed, the number of subcompartments can be substantially increased by adsorption of smaller capsules or nanocapsules onto larger inner capsules, while adsorption can be performed using relatively simple steps: electrostatic self-assembly, hydrogen-bonding, porosity, etc. Using electrostatic self-assembly, the fabrication procedure is comprised of coating both inner and outer particles by polyelectrolytes in their sequential adsorption. Adjusting the relative concentration of small and large particles, one can vary the surface density of the smaller capsules. This structure is associated with ‘raspberry’-like multicompartment capsules. The versatility of this structure is particularly appealing since in this case not only the number of compartments but also their composition can be chosen: for example, micelles, dendrimers, or liposomes.[33,51] In case of capsules, smaller PS or CaCO3 particles coated with polyelectrolyte multilayers can be adsorbed onto the inner compartment. Figure 5 shows smaller PS nanoparticles adsorbed onto CaCO3 templates representing the inner compartment. The outer particles labeled with FITC (green color) are attached to larger particles containing TRITCdextran (red color) and almost completely covered the surface of the inner colloid (Figure 5a). Figure 5b and c present CLSM fluorescence images of these constructs when CaCO3 is dissolved. The third approach is represented by ‘innercentric’ multicompartment capsules wherein one can incorporate
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Figure 5. Fluorescence CLSM images of pericentric multicompartment structures based on the CaCO3 inner core (red) and PS nanoparticles in the outer (green): before the inner core dissolution (a). After the inner core dissolution, two different fluorescent channels (b, green and c, red) are shown for the same microcapsule.
a number of smaller particles or capsules inside a bigger compartment (Figure 1c). In some way, the initial step of this structure is similar to that of the ‘concentric’ capsule. The simplest constructs of ‘innercentric’ (cherry-like) and ‘concentric’ (one ring onion-like) models are in fact identical; smaller CaCO3 particles can be readily put inside particles or capsules (Figure 1a and c). This structure is associated with a ‘grape’-like multicompartment construct. It can be noted that, although some asymmetry may be constructed in the case of pericentric and innercentric structures, all of the above capsules are isotropic, in general. Anisotropic constructs are depicted in the fourth approach, which demonstrates ‘acentric’ multicompartment capsules. A prototype of this structure is realized by direct synthesis of CaCO3 templates (Figure 1d), resulting in an ‘acorn’-like multicompartment capsule. The acentric approach is different from the three previously described
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structures in that it is an anisotropic construct. Here, the structure is realized as a by-product of the CaCO3 making reaction, Figure 6. Although anisotropic capsules are based here on anisotropic particles produced as a by-product of CaCO3 synthesis, other approaches to obtain anisotropic particles can be used. For example, Velev et al. introduced anisotropic particles of different shapes, that were of uniform porosity, monodisperse in size, and highly structured,[52] while Paunov and co-workers reported on the fabrication of anisotropic microcapsules produced by using alternating layers of nano-cotton fibers and oppositely charged polyelectrolytes over anisotropic inorganic templates.[53] Recently, Yu et al. obtained anisotropic polymer particles by protrusion of the PS cores. In that work, polyelectrolyte-coated PS particles were incubated in water/tetrahydrofuran mixtures.[54] Alternative ways of multicompartmentalization were presented by Bhaskar et al. who showed the formation of multicompartmental microcylinders and microdisks, which may play an important role in the development of next-generation biomaterials with precisely designable physical and chemical properties.[55,56] One particularly important area of multicompartmentalization is the simultaneous delivery of small and large molecules. Here, polyelectrolyte multilayer capsules possess a drawback since they can not hold small molecules well (
