Control in Mineralization by the Polysaccharide-Coated Liposome via

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Control in Mineralization by the Polysaccharide-Coated Liposome via the Counter-Diffusion of Ions Yuuka Fukui and Keiji Fujimoto* Center for Chemical Biology, School of Fundamental Science and Technology, Graduate School of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, 223-8522, Japan

bS Supporting Information ABSTRACT: We created an organic inorganic hybrid nanocapsule by utilizing polysaccharide-coated liposomes as a reaction site for the deposition of calcium phosphate (CaP). Phosphate ions were encapsulated in a liposome, followed by the layer-by-layer deposition of chitosan (CHI), dextran sulfate (DXS), or DNA onto the liposome surface. Calcium ions were added to an aqueous suspension of the phosphate ion-incorporated nanocapsules to prepare the nanocapsules that provide a variety of walls for the counter-diffusion of ions and the surface for CaP deposition. As a result, control in biomineralization, such as thickness and crystal properties, over the nanocapsules was achieved by tuning the counter-diffusion of the calcium ions and the phosphate ions through the capsule wall and the surface chemical composition of nanocapsules. Furthermore, we carried out DNA adsorption onto CaP-coated liposomes. DNA was releasable from the nanocapsules because of the dissolution of CaP under acidic conditions. KEYWORDS: liposome, polysaccharide, DNA, calcium phosphate, nanocapsule, layer-by-layer deposition, organic inorganic hybrid

’ INTRODUCTION The development of organic inorganic hybrid materials is of great interest in a variety of fields. Numerous studies on the preparation of hybrid nanoparticles have been reported, because the combined properties of organic and inorganic materials and their synergistic effects are expected to be one of leading candidates of functional materials, which can be applied in diverse fields, including drug or gene delivery systems and biological analysis, such as bioimaging and functional materials science (e.g., semiconductors and photocatalysts). Polymeric particles, micelles, and biopolymers such as DNA and proteins were utilized to obtain their complexes with inorganic compounds such as quantum dots,1 titanium oxide (TiO2),2 4 calcium phosphate (CaP),5 12 and iron oxide (magnetite).13 These approaches can be expected to allow for controlling size distributions, improving dispersion stability, and enhancing biocompatibility by well-organizing the composites of organic and inorganic materials. Here, we focused on CaP as the inorganic component, which is the main constituent of hard tissues in vertebrates, such as bones and teeth, and is the most abundant inorganic material in living organisms. In terms of materials design, the pH-dependent dissolution of CaP is advantageous to render pH-responsivity to carriers for drug or gene delivery. There have been extensive studies on synthesizing hybrid nanocapsules of CaP using polymer gels,12 micelles,10 and liposomes14 19 as templates for mineralization. Crystal properties of CaP such as crystal structures in morphology and thickness of CaP were controllable by r 2011 American Chemical Society

tuning the reaction conditions such as pH,20 temperature, reaction time,15 and addition of capping molecules.14 We have previously reported biomaterial-derived nanocapsules by the layer-by-layer (LbL) deposition of polypeptides or polysaccharides onto negatively charged liposomes.21,22 The LbL method is a versatile and flexible technique to fabricate polyelectrolyte multilayer films through sequential deposition, mainly as a result of the electrostatic attraction between oppositely charged polyelectrolytes.23,24 It is therefore easy to obtain various nanocapsules possessing a variety of capsule walls composed of different types and combinations of polysaccharides. We here intended to control the production of CaP using these nanocapsules as a template for the reaction of calcium ions and phosphate ions. Some researchers have reported that the deposition of CaP over the liposomal surface was often conducted by adding both calcium and phosphate ions to the outer water phase and then controlling reaction conditions such as pH and temperature.14 16 In biological systems, mineralization found in nacre and bone is highly controlled within the closed compartments, such as vesicles and insoluble extracellular organic matrix under moderate conditions, resulting in the formation of hierarchical architectures that provide multifunctional properties25,26 Our approach is based on the concept inspired by such biomineralization as described in Scheme 1. To enable the spatial control of CaP formation over the capsule surface, calcium ions and phosphate ions are compartmentalized into the outer phase Received: April 28, 2011 Revised: August 3, 2011 Published: October 07, 2011 4701

dx.doi.org/10.1021/cm201211n | Chem. Mater. 2011, 23, 4701–4708

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Scheme 1. Preparation of Organic Inorganic Hybrid Nanocapsules via the Counter-Diffusion of Ions Across the Capsule Wall of Polysaccharide-Coated Liposomes (Liponano-Capsules). Phosphate Ions Are Encapsulated Inside a Negatively-Charged Liposomea,b,c

a

Layer-by-layer depositions of polysaccharides are carried out over the liposomal surface, followed by the addition of calcium ions. Here, the orange and green chains represent a cationic polysaccharide, such as chitosan, and anionic polysaccharides, such as dextran sulphate and DNA, respectively. b Calcium ions are attracted to and concentrated around the negatively-charged surface of polysaccharide-coated liposome. Both calcium ions and phosphate ions diffuse through the capsule wall of nanocapsules. c The control in the counter-diffusion of ions and surface chemical composition of polysaccharide-coated nanocapsules enables the spatial control in CaP formation over the capsule surface and, hence, the formation of the organic inorganic hybrid nanocapsule.

and the inner cavity of the capsule, respectively. Phosphate ions and calcium ions would mutually diffuse between inside and outside the capsule wall, respectively, and thereby, it is expected that they could be captured by ionic functional groups of polymeric layers to form CaP selectively at the surface of nanocapsules. The inner pH of the liposome was kept below 4 to prevent the precipitation of CaP inside the liposomal cavity. We here intend to control both the rate and the direction of ion diffusion across the capsule wall by depositing the polymer layer over the liposome. Moreover, a “field” suitable for the precipitation of CaP on the capsule wall can be created by a diverse selection of polysaccharides in terms of affinity with calcium ions and calcium phosphate crystals. In addition to the counterdiffusion of ions and the surface chemical composition of nanocapsules, by controlling the reaction conditions such as pH and temperature, this system would provide us a simple but elaborate way to control not only the thickness of the CaP layer but also the crystal properties, including crystallinity and crystal structures in the morphology of CaP toward biomineralization. It is expected that the combination of CaP with such nanocapsules would provide a novel hybrid nanocapsule with bioinertness, biocompatibility, and pH-responsivity. In fact, the control in mineralization over the nanocapsules allows versatile materials design for bone compatibility, such as absorption capability and specificity toward their use in bone substitute materials,27 and for bone targeting in drug delivery systems.15 It is also expected that antibone resorptive agents such as alendronate, which is used for osteoporosis, could be displayed on the capsule surface through their affinity for hydroxyapatite (HAp). As an approach of the hybrid nanocapsule to biomedical applications, we also investigated pHtriggered release of DNA from resultant CaP capsules depositing DNA by utilizing pH-dependent dissolution of CaP.

’ EXPERIMENTAL SECTION Materials and Methods. Preparation of Phosphate Ions-Encapsulated Liposomes. The negatively charged liposomes were prepared as follows:21,22 Dimyristoylphosphatidylcholine (DMPC) and dilauroyl phosphatidic acid (DLPA) were dissolved in methanol at a lipid ratio of 0.5:0.5. Removal of methanol was done by rotary evaporation to yield a thin lipid membrane over the inner surface of a roundbottom flask. One mL of 100 mM Na2HPO4/NaH2PO4 phosphate buffered solution was added to the lipid membrane in the flask for obtaining a lipid suspension using a bath-type sonicator at 50 °C. The inner pH of the liposome was kept below 4 to prevent precipitation of CaP inside the liposomal cavity. After three cycles of freezing and thawing, the liposome suspension was extruded 20 times through a membrane with pores of 100 nm at 50 °C using a LipoFast Basic instrument (Avestin Inc., Ontario, Canada). Layer-by-Layer Deposition of Polysaccharides onto Liposomes. The first polyelectrolyte layer was deposited onto negatively charged liposomes from CHI aqueous solution containing 100 mM Na2HPO4/ NaH2PO4.21 CHI solution (0.5 mL, 1600 ppm) was added to 0.5 mL of a liposome suspension (DLPA:DMPC = 0.5:0.5) at a final phospholipid concentration of 0.5 mM. The adsorption was carried out for 30 min at 20 °C and a pH of 4.5, stirring at 700 rpm. Then, excess polyelectrolytes were removed by 10 times repeated ultrafiltration of the suspension using the VIVASPIN filtration membrane (300 kD MW cutoff, Sartorius Stedim Biotech Product, Goettingen, Germany) for 2 min at 1500 rpm and 25 °C. The obtained capsule was referred to as liponano CHI. The second layer was fabricated by the addition of a dextran sulfate (DXS) aqueous solution or a DNA aqueous solution at a final concentration of 500 ppm, to the suspension of liponano CHI. The obtained capsule was referred to as liponano CHI DXS or liponano CHI DNA. For instance, the outer water phase of each nanocapsule was exchanged by ultrafiltration to 10 mM HEPES (pH = 7.0) or 10 mM CHES buffer (pH = 10.0). 4702

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Release of Phosphate Ions from the Liposome and Liponano CHI DNA. A suspension (800 μL) of liposomes or liponano CHI DNA (phospholipid concentration of 1 mM), in which phosphate ions were incorporated, was poured into a dialysis unit (QuixSep Micro Dialyzer, Membrane Filtration Products, Inc., Seguin, TX) and dialyzed for 4 d at 37 °C and pH = 7.0 or pH = 10.0. The amount of phosphate ions released from each sample was measured by colorimetric analysis. Precipitation of CaP over the Polysaccharide-Coated Liposomes. A suspension of liposomes (500 μL) at a phospholipid concentration of 0.125 mM, in which phosphate ions were encapsulated, was added dropwise into 500 μL of CaCl2 aqueous solution with stirring at 700 rpm, and the reaction was undertaken for 1 or 18 h at 37 °C and a pH of 7.0 or 10.0. To inhibit the formation of CaP crystals in a bulk solution, the concentration of related ions was adjusted to keep them below the solubility product (Ksp) of HAp [Ca10(PO4)6OH2], which has the lowest Ksp among all of the calcium phosphate crystals.28 The remaining ions were removed by 10 times repeated ultrafiltration of the suspension using VIVASPIN filtration membrane (300 kD MW cutoff, Sartorius Stedim Biotech Product, Goetingen, Germany). Deposition of CaP using liponano CHI, liponano CHI DXS, and liponano CHI DNA as templates was also performed by the same procedure as deposition using bare liposomes. The morphology and crystal structure of produced CaP were observed with a field emission transmission electron microscope (FE-TEM). The samples were prepared by depositing an aqueous suspension of nanocapsules upon a carbon-coated copper grid, followed by air-drying for 5 min. Infrared spectra of the CaP-deposited nanocapsules were obtained in the form of KBr discs using an FT-IR spectrometer (ALPHA, Bruker Optik GmbH, Germany). Precipitates produced over each nanocapsule were also dissolved at a pH of 3 for 2 h, and calcium ions were collected by ultracentrifugal separation (70 000 rpm, 4 °C, 1 h), followed by membrane filtration (cellulose acetate 0.2 μm ADVANTEC, Toyo Roshi, Japan). The concentration of calcium ions was measured by ion-exchange chromatography (Prominence HIC-SP, Shimadzu, Japan). Release of DNA from the Surface of CaP-Coated Liposomes upon Dissolution of CaP. CaCl2 and Na2HPO4/NaH2PO4 were used as ionic species, and CaP deposition on the liposome was carried out at 37 °C. A 1.0 mM lipo CaP suspension (500 μL) was added to 500 μL of 250 ppm TRITC (tetramethylrhodamine-5-(and 6)-isothiocyanate) labeled DNA solution, and the mixture was stirred for 1 h to produce the DNAdeposited capsule (lipo CaP DNA). After unbound DNA was removed by centrifugal separation (15 000 rpm, 4 °C, 1 h), green fluorescence from FITC (fluorescein isothiocyanate) dextran encapsulated inside lipo CaP and red fluorescence from TRITC-labeled DNA were observed by confocal scanning laser microscopy to confirm DNA adsorption onto the lipo CaP. The obtained lipo CaP DNA was redispersed in 1 mL of 10 mM HEPES with a pH of 7.0 or in 10 mM MES with a pH of 4.0 and incubated for 1 and 17 h. Desorbed DNA was separated and collected in the supernatant solution by centrifugal separation (15 000 rpm, 4 °C, 1 h), and the amount of DNA was determined by measuring TRITC fluorescence (λex/λem = 541/572).

’ RESULTS AND DISCUSSION The resultant liposome was unilamellar, with the diameter of approximately 100 nm, and possessed a highly negative charge. In the LbL deposition of various polysaccharides onto the liposomal template, chitosan (CHI) was chosen as the first polyelectrolyte layer, and the obtained nanocapsule was referred to as liponano CHI. As the second polyelectrolyte layer, dextran sulfate (DXS) or DNA was deposited onto liponano CHI, and the obtained nanocapsules were referred to as liponano CHI DXS and liponano CHI DNA, respectively. The covering of the

Figure 1. Transmission electron microscopy images of hybrid nanocapsules after the deposition of CaP over the liposome, liponano CHI, liponano CHI DXS, and liponano CHI DNA at pH valuses of 7.0 and 10.0. Each reaction was carried out at 37 °C for 1 h using CaCl2 and Na2HPO4/NaH2PO4 as ion species.

Figure 2. Release of phosphate ions from the liposome and the liponano CHI DNA at 37 °C and a pH of 7.0 or a pH of 10.0.

liposomal surface with CHI, DXS, and DNA by LbL deposition was confirmed by the change in their electrophoretic mobilities.21 We investigated several key factors in the formation of CaP over the nanocapsules, which are described as follows: (a) the type of nanocapsule as a template for reaction and crystal formation, (b) pH, (c) temperature, (d) reaction time. These factors concertedly have effects on the rates of reaction and ion diffusion, hence, allowing us to control the crystal properties of the CaP wall, such as crystal polymorphs, thickness, and crystallinity. The morphology and crystal structure of the produced CaP were observed with a field emission transmission electron microscope (FE-TEM). At both a pH of 7.0 and a pH of 10.0, precipitates were produced selectively at the surface of almost all of the liposomes to form hybrid capsule walls, as shown in Figure 1 and Figure S1 in the Supporting Information, whereas some were observed as aggregates at pH = 10.0, as a result of the formation of debris at nearby liposomal surfaces. As for sugar liponano-capsules, however, solid particles or large aggregates were formed, and hybrid capsule walls were scarcely observed. We previously reported that the release of fluorescent substances from liposomes was suppressed by the deposition of polysaccharides onto their surfaces20 and this is in good accordance with the release of phosphate ions (Figure 2). The amount of phosphate ions released from liponano CHI DNA was significantly low, at both a pH of 7.0 and a pH of 10.0, compared 4703


Chemistry of Materials to that of bare liposomes. This fact strongly indicates that the release of phosphate ions from the inner cavity of sugar liponanocapsules was too slow to provide sufficient ions for formation of a nucleus and further crystal growth at the surface. In other words, the release of phosphate ions is controllable by the capsule wall. It is well-known that pH is a key factor to control crystal formation and growth of CaP. In practice, HAp is allowed to be formed above pH = 7.0 because of its low value of Ksp. As can be seen in Figure 1, precipitation over the surface of liponano CHI DNA was achieved by the increasing the pH to 10. All of the observed hybrid nanoparticles showed a hollow structure. Rupture of some capsules was also observed, which probably took place upon the precipitation process. At pH = 10, hydroxide ions sufficient for the formation of HAp were readily supplied, although the release of phosphate ions was suppressed by the capsule wall. Therefore, mineralization on the surface of liponano CHI DNA was facilitated to form the hybrid nanocapsule (Figure 1). Furthermore, another important factor in this system is a “site” suitable for CaP deposition (e.g., the affinity for calcium ions), which can be provided by functional moieties on the capsule surface. There have been numerous studies on the effect of organic or polymeric compounds on CaP growth from aqueous media, leading to bioinspired and biomimetic mineralization of CaP.29 31 Compounds containing functional groups such as phosphate and carboxyl are implicated in the adsorption of related ions and further CaP crystallization. Carboxyl groups of collagen are supposed to be responsible for nucleation of HAp on collagen from a simulated body fluid (SBF). In our system, phosphate groups displayed at the surface of the liposome and the liponano CHI DNA are expected to function as more efficient nucleating sites for CaP than the amine groups of CHI and the sulfate groups of DXS. These overall suggest that pH in the external solution and the surface moiety of nanocapsules are important to control nucleation and growth of CaP for the formation of organic inorganic hybrid capsule walls. Elemental analysis was performed by using an energy dispersive X-ray spectrometer (EDX). As shown in the upper panels of Figure 3, the peaks of Ca and P could be observed in the EDX spectra of lipo CaP and liponano CHI DNA CaP

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produced at 37 °C and a pH of 10, whereas bare liposomes and liponano CHI DNA exhibited no peak of Ca (data not shown). This indicates that calcium phosphate was precipitated over the capsule surface. Selected area electron diffraction (SAED) analysis of magnified areas was also conducted to analyze the crystallinity of produced CaP crystals. The lower panels in Figure 3 shows that electron diffraction rings or spots were not observed in the electron diffraction pattern of lipo CaP, indicating that the precipitated CaP was amorphous. On the other hand, a few

Figure 4. FT-IR spectra of liposome (a), liponano CHI DNA (b), lipo CaP (c) and liponano CHI DNA CaP (d). The CaP precipitation was carried out at 37 °C for 1 h using CaCl2 and Na2HPO4/ NaH2PO4 as ion species.

Figure 3. Energy dispersive X-ray analysis spectra (upper) and selected area electron diffraction images (lower) of lipo CaP and liponano CHI DNA CaP produced at 37 °C and pH = 10. 4704



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Figure 5. Transmission electron microscopy observation at high magnification of a hybrid capsule wall produced on a liposome at 37 °C and pH = 10 (left) and on a liponano CHI DNA at 37 °C and pH = 10 (right).

spots appeared in the electron diffraction pattern of liponano CHI DNA CaP, suggesting the crystalline structure of calcium phosphate. FT-IR analysis was carried out to investigate the presence of calcium phosphate and its crystal property in the hybrid nanocapsules. Parts a and b of Figure 4 show the IR spectra of the liposome and the liponano CHI DNA where no characteristic peak of calcium phosphate was observed. As shown in Figure 4c, the spectrum of lipo CaP shows unresolved peaks at 1073 cm 1 (stretching vibration of PO43 ) and 576 cm 1 (deformation vibration of PO43 ), indicating low crystallinity of calcium phosphate.32 In the spectrum of liponano CHI DNA CaP, the peak splitting at the regions of 1050 1100 cm 1 (stretching vibration of PO43 ) and 540 600 cm 1 (deformation vibration of PO43 ) was observed (Figure 4d). This indicates the presence of partially crystalline HAp.32 These correlate well with the results from SAED analysis. There are two reasons why the CaP crystallinity of liponano CHI DNA CaP is higher than that of lipo CaP. First, the release rate of phosphate ions from liponano CHI DNA was lower than that from bare liposomes. This slow release probably led to the gradual growth of CaP, resulting in an increase in crystallinity. Another reason is that the phosphate group in DNA might act as a template suitable for an epitaxial nucleation and growth of CaP because its arrangement was in good conformity to the lattice spacing on the top face of the CaP crystal. For instance, DNA is known to strongly bind with hydroxyapatite by electrostatic interaction or formation of chelating complex with calcium ions on its surface. A hydroxyapatite column is commercially utilized for purification or isolation of DNA.33 Therefore, these suggest that the polysaccharide wall of the sugar liponano-capsules was a good template for CaP crystal nucleation and growth. Lipo CaP (37 °C and pH = 10) was observed by field emission scanning electron microscopy (FE-SEM). Bare liposomes did not maintain their spherical shape, whereas a large majority of lipo CaP retained their original shape even after a natural drying process, suggesting that a rigid CaP layer was formed on the capsule wall (see Figure S2 in the Supporting Information). The cross section of liponano CHI DNA was examined by a TEM at high magnification (Figure 5). The lipid membrane wall was observed as a white circle at the middle of the capsule, and a double-lined dark layer could be observed, strongly suggesting that thin layers of CaP were deposited both at the outer and at the inner sides of the capsule wall (Figure 5 (right)). On the other hand, the surface of the bare liposomes was covered

Figure 6. Effect of incubation time on the formation of CaP crystals using the liposome and the liponano CHI DNA as a template. Each reaction was carried out at pH = 7.0 and pH = 10.0 for 18 h, using CaCl2 and Na2HPO4/NaH2PO4 as ion species. The images were observed by a FE-TEM.

Figure 7. Desorption of DNA at pH = 4.0 and pH = 7.0 from lipo CaP as a function of time.

with the thick inorganic layer as shown in Figure 5 (left). The formation of CaP in the inner phase of liponano CHI DNA strongly indicates the gradual and mutual transfer of each ion across the polysaccharide-deposited capsule wall. On the contrary, diffusion of phosphate ions from the liposome took place so rapidly that precipitation occurred only at the outer side of the membrane before allowing calcium ions to diffuse inside. This once again supports that the CaP deposition was governed by the rate and the mode of ion diffusion across the capsule wall. It is known that CaP formation proceeds quickly at high temperatures;33 therefore, we investigated the effect of the reaction temperature particularly on the CaP deposition over the surfaces of the liposome and the liponano CHI DNA (see Figure S3 in the Supporting Information). When the reaction temperature was raised from 37 to 60 °C, all of the lipo CaP changed from a spherical shape to large aggregates or a needlelike shape, irrespective of pH. For liponano CHI DNA, hybrid nanocapsules could only be obtained at 37 °C and a pH of 10 (Figure 1), whereas solid hybrid particles were obtained by the 4705



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Scheme 2. Schematic Representation of Controlling in Mineralization over the Surface of Nanocapsules by Tuning the Rate and Direction of Ion Diffusion, Surface Functional Groups and the Reaction Conditions

formation of CaP inside all nanocapsules at 60 °C and a pH of 10. These suggest that temperature has an influence on the ion transfer across the capsule wall. By increasing the temperature, the protons can be released from the nanocapsule, and thereby, the internal pH of the nanocapsule rapidly increases, resulting in nucleation and growth of CaP inside the nanocapsule. This is why the formation of large hybrid aggregates or solid particles was observed at high temperatures. We intended to control the thickness of CaP capsule wall by prolonging the reaction time from 1 to 18 h. The results of the TEM observation are shown in Figure 6. When the liposome was used as a template, hybrid nanocapsules were obtained by depositing CaP selectively at the surface after 1 h-incubation as shown in Figure 1, and eventually, after 18 h-incubation, they were all ruptured at a pH of 7.0 and large needlelike crystals were formed at a pH of 10. On the other hand, a well-defined CaP capsule wall could be observed over some of the liponano CHI DNA, and the wall thickness appeared to increase both at a pH of 7.0 and at a pH of 10.0. It is thought that this morphological difference between a lipo CaP and a liponano CHI DNA CaP should be attributed to the rate of the ion transfer across the capsule wall, as well as the temperature effect described above. Therefore, it is concluded that a sustained release of ions from liponano CHI DNA allowed a gradual precipitation of CaP, which led to control in the thickness of capsule wall. This emphasizes that the control in the rate and mode of ion diffusion by LbL deposition of biopolymers onto the liposome is important to optimize crystal formation with respect to the site and crystal properties. This is the concept of the bioinspired mineralization, which is enabled by the use of liponano-capsules. In addition, CaP crystals are known to be able to dissolve by decreasing pH. The local pH around the ruffled border of osteoclasts34 during bone remodeling and the endosomal pH of cells are known to be acidic. It can be also expected that rapid

and smooth degradation of the inorganic layer could allow prompt release of DNA upon the pH decrease when nanocapsules were internalized into the phagolysosome.6 Therefore, to produce the DNA-releasable capsule in response to pH, we intended to bind DNA on the surface of the CaP wall. DNA deposition was proven from the fluorescence observation by confocal scanning laser microscopy. Both green fluorescence from FITC-dextran encapsulated inside lipo CaP and red fluorescence from TRITC-labeled DNA, which was expected to be deposited on the surface of lipo CaP, could be observed at the site of the nanocapsules (data not shown). The adsorption amount of DNA onto the surface of lipo CaP was measured to be 23.8 ng/cm2. As can be seen in Figure 7, DNA release was slow after the initial burst at pH = 7.0, whereas DNA was abruptly released from lipo CaP DNA and the release was almost 90% within 1 h at pH = 4.0. As expected, the release kinetics of DNA correlated well with the dissolution curve for the CaP layer. CaP dissolution took place much more rapidly at pH = 4.0 than at pH = 7.0, and the dissolution leveled off within around half an hour (see Figure S4 in the Supporting Information). This is presumably due to the pH-dependent solubility product of CaP, which is known to increase with decreasing pH. In addition, a dense and rigid inorganic layer could no longer be observed from the TEM image of Lipo CaP after its incubation at pH = 4 (see Figure S6 in the Supporting Information). These results strongly suggest that the release of DNA was triggered by the solubilization of the CaP layer. In terms of its practical use in a living body, it is important to investigate the influence of the components of a body fluid such as ions and proteins on CaP dissolution. Thus, we performed a preliminary experiment to investigate how bovine serum albumin (BSA) influenced the dissolution of the CaP layer (see Figure S5 in the Supporting Information). As a result, BSA did not exhibit any significant interference on CaP dissolution at low pH. Such a carrier design will open up the possibility of pH4706


Chemistry of Materials triggered release from the surface of nanocapsules, which is advantageous for the delivery and release of a variety of substances including drugs and genes in the body. In Scheme 2, we summarize control in mineralization by utilizing liponano-capsules. The spatial control of CaP formation over the capsule surface could be controlled by tuning the rate and direction of ion diffusion across the capsule wall, surface functional groups of the capsule wall, and the reaction conditions. Calcium ions and phosphate ions were compartmentalized into the outer phase and the inner cavity, respectively, for the counter-diffusion of each ion across the capsule wall. Mineralization on a bare liposome was governed by the rapid and unidirectional release of phosphate ions, so it was difficult to control site-specificity in the mineralization and the crystallinity of CaP. The slow and mutual diffusion was tunable by the polysaccharide layer of the capsule wall. The surface layer possessing the affinity for calcium ions and calcium phosphate crystals was suitable for the precipitation of CaP. Liponano CHI DNA allowed for the presenting of phosphate moieties on the surface, in addition to the slow release of ions. This resulted in the formation of thin and crystalline CaP and also control in thickness of CaP wall by prolonging the reaction time. We are currently studying the optimization of the CaP wall toward the bone-targeted delivery system and the gene delivery system.

’ CONCLUSION We demonstrated the generation of organic inorganic hybrid nanocapsules by the deposition of calcium phosphate (CaP) over the surface of liposome or polysaccharide-coated liponanocapsules. The selection of the nanocapsule as a template and the tuning in of the reaction conditions are important for controlling the rate of ion transfer via the capsule wall and, thereby, CaP formation, and the deposition site and the crystal property were also controllable by using nanocapsules as a template. TEM observations showed that the wall of CaP was formed selectively over the surfaces of bare liposomes and DNAcoated nanocapsules through the mutual diffusion of calcium ions and phosphate ions and their condensation on the capsule surface, whereas no CaP formation occurred on the surfaces of CHI-coated and DXS-coated nanocapsules. From the results of electron diffraction and infrared analysis, the CaP wall over the DNA-coated nanocapsules showed higher crystalline composition than did that over the surface of liposome. Lipo CaP DNA was prepared by adsorption of DNA onto the lipo CaP, and the pH-triggered release of DNA could be observed as a result of the dissolution of CaP at low pH. ’ ASSOCIATED CONTENT

bS

Supporting Information. TEM images of lipo CaP; SEM image of lipo CaP; effect of pH and temperature on the formation of CaP crystals using liposome and liponano CHI DNA as a template; effect of pH on the dissolution kinetics of CaP deposited over the liposome; effect of presence of model protein, bovine serum albumin (BSA), on dissolution of CaP coated over the liposome, TEM image of Lipo CaP after its incubation at pH 4. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

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