ARTICLE pubs.acs.org/Langmuir
Preparation and Assembly of Poly(arginine)-Coated Liposomes To Create a Free-Standing Bioscaffold Saika Yamamoto, Yuuka Fukui, Sachiko Kaihara, and Keiji Fujimoto* The 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 a free-standing membrane as a novel bioscaffold through the assembly of polymer-coated liposomes. Polyarginine (PArg) possessing a cell-penetrating activity was used to form the polymer layer onto a negatively charged liposome (lipo-PArg). The capsule wall of PArg over liposomes made it possible to improve the mechanical property of capsules and to display deoxyribonucleic acid (DNA) over the vesicle surface through the electrostatic attraction (lipo-PArgDNA). The release rates of a fluorescent probe encapsulated in lipoPArg and lipo-PArgDNA were tunable by the number of polymeric layers of the capsule walls. To investigate the cell-membrane permeability of lipo-PArgDNA, polymer-coated liposomes were incubated with human umbilical vein endothelial cells (HUVECs) at 4 °C. It was found that lipo-PArg underwent a significant cellular uptake, whereas bare liposomes and liposomes modified with chitosan were incapable of overcoming the plasma membrane barrier. To prepare a free-standing membrane composed of polymercoated liposomes, a suspension of lipo-PArg-DNA was cast over a mesh hole and dried up. SEM observation revealed that a freestanding membrane was obtained through drying-mediated assembly process without rupturing polymer-coated liposomes inside the membrane. On the other hand, it was not possible to obtain a complete membrane from a mixture of lipo-PArg and DNA. In summary, lipo-PArgDNA capsules possess versatile functions as a drug carrier, and their assembly enables us to create a freestanding membrane applicable as a bioscaffold.
’ INTRODUCTION Recently, intensive research efforts are focused on the fabrication of functional biomaterials via organization of nanoparticles.1,2 Assembly of nanoparticles would provide a membrane with properties different from bulk materials, such as a high specific surface area, a lightweight and flexible body, a large storage capability, and a nanoporous structure. Nanoparticles are applied for a mechanochemical transducer to control physical and chemical properties of biointerfaces and an adhesive plaster for wound dressing, as the structural hierarchies suit well with living structures associated with cells and ECM components.3 In the tissue-engineering, biodegradable and bioderived materials are being used as scaffolds with a variety of structures for cell growth and tissue regeneration.4 Bioderived polymers such as peptides, proteins, and polysaccharides are regarded as a potential material to produce scaffolds due to their biocompatibility and low cytotoxicity. Recent interests are shifted to the utilization of more safe and bioinert scaffolds assembled from bioderived materials and the development of sophisticated tissue regenerative methods provided by introduction of functions such as a controlled release of growth factors and genes.5 It is expected that a membrane assembled from such bioderived polymers will be not only biocompatible but also elastic and robust, and will have an ability to immobilize and display bioactive molecules such as peptides and DNA, which will be applicable for cell culture and r 2011 American Chemical Society
gene transfection. In addition, hollow materials are also expected to be a more potential component (nanocapsule) for construction of scaffolds because a variety of substances can be encapsulated into the capsule wall and the inner cavity. Liposomes are lipid capsules composed of spherical lipid bilayers, which have been utilized as delivery vehicles for drugs and genes due to their ability to encapsulate and deliver active drugs as well as their low toxicity and small size.6,7 A unique technique to create a capsule wall over the liposome surface has been accomplished, which was based on the layer-by-layer deposition of polypeptides and polysaccharides onto the liposomal surface.810 This modification to create the polymer-coated liposome has allowed for providing the robustness of capsules and controlling of the release of encapsulated substances.9,11 It is thought that these features of capsules are advantageous to production of scaffolds by assembling polymer-coated liposomes. In fact, there is an increasing demand for techniques using soft materials because they can be further translated into flexible devices with a variety of functions.12 We think that polymercoated liposomes are one of the promising materials because they can produce different capsule walls by the layer-by-layer Received: April 23, 2011 Revised: June 9, 2011 Published: June 17, 2011 9576
dx.doi.org/10.1021/la201500b | Langmuir 2011, 27, 9576–9582
Langmuir
ARTICLE
Figure 1. Preparation of polymer-coated liposomes and fabrication of free-standing membranes from polymer-coated liposomes and DNA by dryingmediated assembly over the microholes of the mesh grid. Polymer-coated liposomes were prepared by layer-by-layer deposition of polyarginine and DNA onto the liposome (lipo-PArgDNA).
deposition of a variety of polymers. Here, we focused on polyarginine (PArg) because arginine oligopeptides are well-known to possess a cell-penetrating activity, which serves a function in delivering DNA and liposomes.7,1318 We expected that surface modification by PArg would provide the bioactive polymer-coated liposome (lipo-PArg) with possibilities to overcome the permeability barrier of the plasma membrane and transfect substances into the cytosol.19,20 Moreover, the obtained free-standing membrane assembled from lipo-PArgDNA would be expected to function as a flexible substrate with the cell-penetrating activity. The fabrication of free-standing membranes has been achieved by a microhole-confined assembly of nanoparticles to stabilize the structure on drying.21 In such a bottom-up assembly, DNA is often utilized as a dry ligand to produce a free-standing nanosheet of nanoparticles.2224 It has also been reported that the freestanding membrane was constructed from gold nanoparticles modified with DNA via drying-mediated process, and this nonspecific DNADNA interaction was strong enough to lift free-standing nanoparticle superlattices in a dried state.12,21,25 From the viewpoints described above, we here introduce a concept to create a bioscaffold through the assembly of liposomes coated with PArg and DNA as schematically drawn in Figure 1. We first prepared a polymeric capsule wall by adsorption of PArg and DNA onto the liposome surface (lipo-PArg and lipo-PArgDNA) and measured surface potentials and observed surface morphologies of polymer-coated liposomes. The surfactant Triton X-100 was added to the liposomal suspension to investigate a resistance of the capsule wall to solubilization. The release rate of a fluorescent probe was examined to clarify the integrity of the capsule wall. Cellular uptake of the capsules was studied using human umbilical vein endothelial cells (HUVECs) to evaluate the cell penetrating activity of PArg, which was displayed on the liposome surface. A free-standing membrane was prepared to obtain a bioderived scaffold (bioscaffold) by drying-mediated assembly from a suspension of lipo-PArgDNA, and the resultant membrane was observed by scanning electron microscopy. The bioscaffold will be tunable in elasticity and topography by controlling the assembly process and selecting capsule materials. It is expected that such a bioscaffold of lipoPArgDNA will have capabilities of immobilization of bioactive
molecules such as peptides and DNA on the surface and of their efficient transport across the cell membrane by the surfacedisplayed PArg. In addition, the bioscaffold will provide a lot of inner cavities to encapsulate a variety of substances, such as drugs and growth factors, and will also allow their release from the capsules.
’ MATERIALS AND METHODS 2.1. Materials. Dilauroyl phosphatidic acid (DLPA) and dimyristoyl phosphatidylcholine (DMPC) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) and the Nippon Oil and Fats Co. (Tokyo, Japan), respectively. Poly-L-arginine hydrochloride (PArg, MW 15 000 70 000) and 1-hydroxypyrene-3,6,8-trisulfonic acid (HPTS) were purchased from Sigma-Aldrich Co. (St. Louis, MO). DNA derived from salmon spermary was donated from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Chitosan (CHI, MW 10 000100 000) was purchased from Yaizu Suisankagaku Industry Co., Ltd. (Shizuoka, Japan). Fluorescein isothiocyanate labeled dextran (FITC-dextran, MW 150 000) was purchased from Polysciences Inc. (Warrington, PA). Acridine orange was purchased from Dojindo Laboratories (Kumamoto, Japan). Sulfuric acid was purchased from Junsei Chemical (Tokyo, Japan). The other reagents were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Human umbilical vein endothelial cells (HUVECs) and endothelial growth medium (EGM-2 Bullet Kit) were purchased from Cambrex Co. (New York). All of the chemicals were used as received. The water used in all experiments was prepared in a water purification system (WT-100, Yamato Scientific Inc., Tokyo, Japan) and had a resistivity higher than 18.2 MΩ cm. 2.2. Preparation of Liposomes and Encapsulation of Chemical Substances into Liposomes. The negatively charged liposomes were prepared as follows. DMPC and DLPA were dissolved in methanol at different molar ratios. Removal of methanol was done by rotary evaporation to yield a thin lipid membrane over the inner surface of a round-bottom flask. One milliliter of a HPTS aqueous solution (10.0 mM) or a FITC-Dextran aqueous solution (2.0 g/L) in MES buffers (100 mM, pH5.5) was added to the lipid membrane in the flask, and the membrane was dispersed using a bath-type sonicator at 50 °C. 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 (Avestin Inc., Ontario, Canada). The resultant 9577
dx.doi.org/10.1021/la201500b |Langmuir 2011, 27, 9576–9582
Langmuir
ARTICLE
Table 1. Amount of PArg Adsorbed onto a Liposome and ζ-Potentials of Lipo-PArg Produced at pH 5.5 and 7.4a adsorption/ng cm2
pH
DLPA/DMPC
salt conc./mM
liposome
5.5
0.5/0.5
10
lipo-PArg
5.5
0.5/0.5
10
125
0.5/0.5
100
158
0.1/0.9
100
7.4
ζ-potential/mV 70
78.7
0.5/0.5
10
165
0.5/0.5
100
193
0.1/0.9
100
85.7
47 36 26 16 21 56
a Adsorption was carried out at 500 ppm PArg and 20 °C. The adsorption amount was obtained by measuring unbound PArg in a micro BCA method. ζ-Potentials of lipo-PArg were measured in 10 mM HEPES (pH 7.4).
suspension was purified by gel permeation chromatography followed by ultracentrifugal separation.
2.3. Adsorption of Poly(arginine) Polymer onto Liposomes. PArg was adsorbed onto negatively charged liposomes in MES buffer (10 or 100 mM, pH 5.5) or HEPES buffer (10 mM HEPES, pH 7.4). A 0.5 mL aliquot of PArg solution was added to 0.5 mL of the liposome suspension (DLPA:DMPC = 0.5:0.5 or 0.1:0.9) to adjust the final lipid concentration to 0.38 mM. The adsorption at different concentrations of PArg was carried out for 30 min at 20 °C with stirring at 700 rpm. Then excess PArg was removed by 10 times repeated ultrafiltration of the suspension. The obtained capsule was referred to as lipo-PArg. To estimate the amount of deposited PArg, the concentration of PArg in the extracted supernatant was measured by using a Micro BCA Protein Assay Kit (Pierce, Rockford, IL). Lipo-CHI with the final concentration of CHI at 0.8 g/L was prepared by using CHI in MES buffer with the same procedure. After the ultrasonication for 30 s was repeated 10 times for degradation of DNA, the resultant DNA was added to the suspension of lipoPArg to deposit DNA onto the outer surface of lipo-PArg (lipo-PArg DNA) in the same manner as above. The amount of DNA adsorbed onto the surface of lipo-PArg was determined by an acridine orange assay with the use of calibration curve of the fluorescent intensity of acridine orange. DNA was labeled with TRITC through amide formation by carbodiimide method26 for further experiments.
2.4. Characterization of Liposomes and Polymer-Coated Liposomes. Hydrodynamic diameters of liposomes and lipo-PArg were measured by dynamic light scattering using a photon correlator (PAR-3, Otsuka Elecrtronics, Osaka, Japan). Electrophoretic mobilities of liposomes and lipo-PArg suspended in HEPES buffer (10 mM HEPES, pH 7.4) were measured using a ζ-potential analyzer (ZC-2000, Microtec Co., Ltd., Tokyo, Japan). Release profiles of HPTS loaded into lipo-PArg were traced using the fluorescent assay. Liposomes or lipo-PArg suspension were dialyzed for 52 h against 10 mL of MES buffer (10 mM, pH 5.5) at 25 °C. After a defined time interval, the fluorescence intensity (I) at 512 nm of HPTS released into MES buffer was measured at an excitation wavelength of 413 nm using a spectrofluorometer (FP-6500, JASCO, Japan). Lipo-PArg was lysed by the addition of 1% TritonX-100 aqueous solution (0.10 mL) to 1 mL of the capsule suspension, and the intensity (I0) in the lysate was measured. Release of HPTS was calculated from I and I0 using the following expression. release ð%Þ ¼ I=I0 100
2.5. Interaction of FITC-Dextran-Loaded Polymer-Coated Liposomes with Cells. HUVECs suspended in endothelial growth
medium were seeded at a density of 180 000 cells/mL (5.0 104 cells/ cm2) into each well of a 24-well microplate (Asahi Techno Glass Co., Chiba, Japan) with a cover glass (Matsunami Glass Ind., Osaka, Japan) pretreated by acidic collagen solution (1 mL, 0.5%). HUVECs were cultured in EGM at 37 °C for 3 h in an atmosphere of humidified air
containing 5% CO2. Next, FITC-dextran-loaded liposome, lipo-PArg and lipo-CHI, and TRITC-loaded lipo-PArgDNA (0.5 mM phospholipids) suspended in 500 μL of phosphate buffered saline (PBS, pH 7.4) with Ca2+ and Mg2+ were added to each well, followed by incubation for 2 h at 37 or 4 °C. The initial fluorescence (I0) of FITC-dextran or TRITC loaded into each capsule was measured by a spectrofluorometer (FP6500, JASCO, Tokyo, Japan). The cells were then washed twice with PBS, fixed with 3% paraformaldehyde for 30 min at 25 °C, and washed twice again with PBS. The liposomecell interaction was examined with a confocal laser scanning fluorescence microscope (Radiance2000, BioRad Laboratories, Inc., CA) equipped with a 60 oil immersion lens. After the cell incubation was performed for 2 h, fluorescence intensities (I) of FITC-dextran and TRITC of the supernatants of each well before washing were measured by the spectrofluorometer. Amounts of liposomes, lipo-PArg, lipo-CHI, lipo-PArgDNA500, and lipo-PArg DNA2000 incorporated into HUVECs were evaluated by subtracting I from I0.
2.6. Assembly of Polymer-Coated Liposomes into FreeStanding Membrane. Free-standing nanoparticle membranes were
fabricated by using a sheet mesh copper grid (127 μm pitch size, Cu127) and sheet mesh stainless grids (127 and 500 μm pitch size, SUS127 and SUS500, respectively) with a sheet thickness of 10 μm as a holey template. The mesh grids were pretreated by immersing in 1 mL of sulfuric acid and washed with water. Next, the grids were treated by plasma irradiation to make them hydrophilic. Lipo-PArgDNA was suspended in 10 mM MES buffer (pH 5.5). A droplet of lipo-PArgDNA (1 mM phospholipids, 100 μL) was added and trapped in individual microholes of a mesh grid. Next, water was evaporated through natural drying under 30 °C for 18 h so as to thin the droplets in the microholes and form a nanocomposite membrane of polymer-coated liposomes and DNA. These membranes were then observed by confocal laser scanning microscopy, field-emission scanning electron microscopy (FE-SEM S-4700, Hitachi, Tokyo, Japan), and TEM.
’ RESULTS AND DISCUSSION The hydrodynamic diameters of liposomes were around 100 nm, the same size as the pore of the extrusion membrane. The surface charge of liposomes exhibited highly negative at pH 5.5 and 7.4 due to the phosphate groups of DLPA, indicating that the surface charge was tunable with DLPA/DMPC ratios and pH of the suspension medium (data not shown). Table 1 shows the adsorption amount of PArg and ζ-potentials of PArg-adsorbed liposome (lipo-PArg) prepared at different salt concentrations, pH values, and DLPA/DMPC ratios. When the adsorption of PArg was carried out in 100 mM HEPES at pH 5.5 and DLPA/ DMPC = 0.5/0.5, the hydrodynamic diameter of lipo-PArg remained monodisperse in size, and the ζ-potential was changed from 70 of the parent liposome to 36 mV. TEM observation revealed that lipo-PArg maintained their spherical shapes without 9578
dx.doi.org/10.1021/la201500b |Langmuir 2011, 27, 9576–9582
Langmuir
ARTICLE
Figure 2. TEM images of lipo-PArg covered with intact DNA (A) and with degraded DNA (B). Scale bars are 0.1 μm. ζ-potentials of lipo-PArgDNA and adsorption amount of degraded DNA onto a lipo-PArg at pH 5.5 and 100 mM (C). The amount of DNA was measured by an acridine orange assay.
the rupturing of the capsule walls (Supporting Information S1). Irrespective of pH, lipo-PArg prepared in 100 mM HEPES possessed the positive surface charge while keeping a spherical shape. The surface charges of lipo-PArg, which was prepared at DLPA/DMPC = 0.1/0.9, were positive despite the pH, whereas the adsorption amount decreased as compared to DLPA/DMPC = 0.5/0.5. This is because the adsorption was governed by the electrostatic interaction between the negatively charged DLPA and the positively charged PArg. In 10 mM HEPES, it is ant icipated that positively charged PArg chains are adsorbed in extended forms onto the negative surface of the liposome. The adsorption amount and the surface charge in 10 mM HEPES (pH 5.5) were similar to those in 100 mM HEPES. However, the surface of lipo-PArg in 10 mM HEPES (pH7.4) exhibited a negative charge, although the adsorption amount of PArg was as high as those in 100 mM. The electrostatic attraction between liposomes and PArg is weaker at pH 7.4 than that at pH 5.5. PArg chains will be adsorbed in the extended form onto the liposome surface at low salt concentrations (10 mM). It is thought that PArg chains could not bend to adapt to the curvature of the lipid membrane, so that a part of each chain would bind to the liposome surface with a strong negative charge. Therefore, we suppose that the negative charges of the lipid membrane were not fully neutralized by adsorption of PArg chains.27 To study the effect of the PArg deposition on the mechanical stability (robustness) of a capsule wall, the scattering intensity of each suspension of liposomes and lipo-PArg was measured with the addition of TritonX-100 as a surfactant (Supporting Information S2). The scattering intensities of a parent liposome suspension dropped markedly as the surfactant was added at fixed intervals, indicating that the lysis of the lipid bilayer was induced by the surfactant. In contrast, a suspension of lipo-PArg maintained its scattering intensity during the addition of the surfactant, indicating relatively high durability of the capsule wall against Triton-X 100. TEM images presented that the vesicle structure of lipo-PArg was kept intact even in the presence of the surfactant. These results implied that the capsule wall produced by depositing polymeric layers serves as a resistant barrier from solubilization of polymer-coated liposomes. DNA deposition was carried out by electrostatic interactions between positively charged lipo-PArg and negatively charged DNA. From a TEM image in Figure 2A, it was found that intact DNA, which was derived from salmon spermary, induced aggregation of polymer-coated liposomes by bridging between the adjacent lipo-PArg with long DNA chains. Deposition of DNA
Figure 3. Percentage release of HPTS from parent liposome and polymer-coated liposomes as a function of time. The release was followed by measuring the fluorescent intensity of HPTS.
fragments, which were degraded by ultrasonication, could lead to suppression of flocculation (Figure 2B). The binding of DNA onto the PArg-coated liposome should be governed by both their electrostatic interaction and the chain stiffness of DNA. In this study, DNA chains were degraded into fragments, which are shorter than their persistence length. Accordingly, we suppose that the binding was not so influenced by the chain stiffness. The ζ-potentials were positive when adsorption was carried out at the DNA concentration of 200 and 500 ppm, whereas the surface possessed the negative charge at 1000 and 2000 ppm (Figure 2C). This suggests that the capsule wall was fully covered with degraded DNA at high concentrations. In addition to the mechanical stability, it is expected that the capsule wall could provide a barrier against the release of incorporated substances from the inner cavity. Thus, the release profiles of the fluorescent probe, HPTS, from polymer-coated liposomes were investigated as compared to a parent liposome at 25 °C. As shown in Figure 3, the amount of HPTS released from the liposome increasingly attained more than 30% of the initial amount of the incorporated HPTS. On the other hand, the releases from lipo-PArg and lipo-PArgDNA were suppressed as low as approximately 10% and 3%, respectively. It is thought that the membrane fluidity was reduced by polymer deposition, leading to a decrease in the permeability of substances via the capsule walls. To investigate cellular uptake of liposomes and polymercoated liposomes, FITC-dextran encapsulated nanocapsules were incubated with HUVECs, and the fluorescence images were taken with the confocal laser scanning microscope, and their internalization was quantitatively evaluated by fluorescent 9579
dx.doi.org/10.1021/la201500b |Langmuir 2011, 27, 9576–9582
Langmuir
ARTICLE
Figure 4. Confocal laser microscopic images of cellular uptake of liposome, lipo-PArg, lipo-CHI, and lipo-PArgDNA500 after 2 h-incubation at 37 °C (top row) and 4 °C (bottom row). FITC-dextran (green) was encapsulated in each nanocapsule. Deposition of DNA onto lipo-PArg was carried out at the DNA concentration of 500 ppm. All scale bars are 10 μm.
analysis. Figure 4 shows the subcellular distribution of nanoparticles after 2 h-incubation with HUVECs at 37 and 4 °C. Chitosan-modified polymer-coated liposome (lipo-CHI) was used to investigate the effect of the type of polymers on cellular uptake. It could be observed that liposomes and polymer-coated liposomes were internalized into the cell at 37 °C. Although it was difficult to identify each liposome as a capsule, polymercoated liposomes covered with a polymer layer seemed to remain a spherical form, including the FITC-dextran. It has been reported that such cell-penetrating peptide transduction occurs in a receptor- and energy-independent manner.28 Therefore, we also carried out experiments at 4 °C to explore an energyindependent endocytosis of liposome and polymer-coated liposomes (lipo-PArg, lipo-CHI, and lipo-PArgDNA). The internalization of bare liposomes was scarcely observed at 4 °C. LipoCHI underwent the binding onto the cell surface rather than the internalization, probably due to the electrostatic attraction. We used lipo-PArg-DNA prepared at the DNA concentration of 500 ppm for deposition (lipo-PArgDNA500), where the surface of lipo-PArg was not completely covered with DNA, with intent to enable uncovered PArg moieties to fulfill the cell-penetrating activity. As we expected, lipo-PArg and lipo-PArgDNA500 were internalized into the cell. It is thought that this was attributed to the cell penetrating activity of PArg. Figure 5 shows the amount of liposomes, lipo-PArg, lipo-CHI, and lipo-PArgDNA incorporated into HUVECs for 2 h at 37 and 4 °C. The cellular uptake of bare liposomes was suppressed by changing the temperature from 37 to 4 °C, whereas the internalization of lipo-PArg was temperature-independent and greater than that of bare liposomes. This indicates that the internalization of lipo-PArg is attributed to the cell-penetrating activity of PArg. Deposition of degraded DNA onto lipo-PArg was carried out at DNA concentrations of 500 and 2000 ppm to prepare lipo-PArgDNA500 and lipo-PArgDNA2000, respectively. Interestingly, lipo-PArg DNA500 and lipo-PArgDNA2000 were internalized even at 4 °C, suggesting the temperature-independent internalization. This indicates that the cell-penetrating activity of PArg remained even after the deposition of DNA on the surface of lipo-PArg. It was also found that the amount of cellular uptake of lipo-PArgDNA
Figure 5. Amounts of liposomes, lipo-PArg, lipo-CHI, lipo-PArg DNA500, and lipo-PArgDNA2000 incorporated into HUVECs after 3 h-incubation at 37 and 4 °C. Deposition of degraded DNA onto lipoPArg was carried out at the final concentration of 500 and 2000 ppm for lipo-PArgDNA500 and lipo-PArgDNA2000, respectively.
decreased with an increase in the amount of deposited DNA. This implies that the surface coverage of DNA over the PArg moieties influences cellular uptake of lipo-PArgDNA. Cellular uptake of lipo-CHI could be observed at 37 °C, whereas its internalization was inhibited at 4 °C. These results indicate that lipo-PArg and lipo-PArgDNA possess the cell-penetrating activity of PArg moieties. Next, we intended to prepare a free-standing membrane assembled from lipo-PArgDNA. The obtained free-standing membrane of lipo-PArgDNA would be expected to function as a flexible substrate with the cell-penetrating activity. The fabrication of free-standing membranes has been achieved by a microhole-confined assembly of nanoparticles to stabilize the structure on drying.21 According to this method, an aqueous suspension of nanocapsules was blotted over a metal mesh with the microholes. Plasma treatment was carried out onto the mesh surface so as to optimize the dewetting and the thinning of the suspension into the microhole. Here, dewetting takes place on the mesh, and the remaining suspension of nanocapsules is trapped into each 9580
dx.doi.org/10.1021/la201500b |Langmuir 2011, 27, 9576–9582
Langmuir
ARTICLE
Figure 6. Confocal laser microscopic images of free-standing membranes formed over the microholes of a mesh copper grid with a 127 μm pitch size. Each free-standing membrane was assembled from bare liposomes (A), lipo-PArg (B), or lipo-PArgDNA (C). A dark fluorescence image of (A) and bright fluorescence images of (B) and (C) correspond to the membrane parts suspended over mesh holes. Deposition of degraded DNA onto lipo-PArg was carried out at the final concentration of 500 ppm. TEM images of the free-standing membrane of lipo-PArgDNA (D and E). All scale bars are 20 μm.
individual microhole as water evaporates from the suspension. The suspensions of bare liposomes and polymer-coated liposomes (lipo-PArg and lipo-PArgDNA) were used to prepare the free-standing membranes over the microholes of a mesh copper grid with a 127 μm pitch size (Cu127). We could not observe membrane-like structures for bare liposomes. Fluorescence microscopic observation revealed that lipo-PArg was assembled into partially ruptured membranes and the mesh surface was stained with released FITC-dextran (Figure 6A and B). FITCdextran with a large molecular weight will only leak if substantial defects are present. DNA is often utilized as a dry ligand to produce a free-standing nanosheet of nanoparticles.2224 Therefore, we expected that the DNA corona at the outermost surface of lipo-PArgDNA makes contact with each other and deforms through nonspecific DNADNA interactions upon drying, tightly linking lipo-PArgDNA to form a compliant free-standing membrane. As a result, we could observe that the free-standing membrane was obtained by assembling lipo-PArgDNA and encapsulated FITC-dextran was maintained in the capsules (Figure 6CE). This definitely indicates that deposited DNA worked well as a dry ligand for DNA-controlled assembly of nanocapsules through noncovalent attachment of DNA.12,21,22 Furthermore, the DNA density on lipo-PArgDNA was a key factor to obtaining free-standing membrane as a higher concentration of DNA for deposition onto lipo-PArg was required to obtain complete free-standing membranes for larger microholes (Figure 7A). Adsorption of DNA at the final concentration of 2000 ppm onto lipo-PArg was required to form a free-standing membrane over the microholes of a mesh stainless grid with a 500 μm pitch size (SUS500), whereas 500 ppm of DNA was enough to form an intact membrane in the microholes of a mesh stainless grid with a 250 μm pitch size (SUS250). Cross-sectional
Figure 7. Confocal laser microscopic image (A) and SEM image (B) of a free-standing membrane fabricated with a mesh SUS grid with a 500 μm pitch size. Dark fluorescence images correspond to the membrane parts suspended over mesh holes. The free-standing membrane was constructed with lipo-PArgDNA. Deposition of degraded DNA onto lipo-PArg was carried out at the final concentration of 2000 ppm.
fluorescence images of the free-standing membrane were taken with a confocal laser scanning microscope (Supporting Information S3). It is noteworthy that the obtained free-standing membrane was 23 μm in thickness and assembled from polymercoated liposomes. Morphological observations by SEM also revealed that lipo-PArgDNA found in the membrane maintained the spherical shapes without rupturing of polymer-coated liposomes (Figure 7B). The fact that these free-standing membranes maintained their structures even under vacuum states proved that these membranes possessed both robustness and flexibility. Such highly structured assemblage of lipo-PArgDNA holds a great potential for the utilization as a functional bioscaffold 9581
dx.doi.org/10.1021/la201500b |Langmuir 2011, 27, 9576–9582
Langmuir in drug delivery and tissue engineering, which can exhibit a synergistic effect in drug release from capsules and cell-penetrating provided by the surface-displayed PArg. In addition, this would be expected to be one of safe and bioinert scaffolds in tissue engineering by functionalization such as the controlled release of drugs and bioactive agents such as growth factors and genes.
’ CONCLUSION PArg deposition over a liposome surface (lipo-PArg) made it possible to improve the mechanical property of capsules and to display deoxyribonucleic acid (DNA) over the capsule surface (lipo-PArgDNA). The release of an encapsulated fluorescent probe from these polymer-coated liposomes was suppressed by the polymeric layers of the capsule walls. PArg-displayed polymercoated liposomes exhibited cell-membrane permeability even under nonmetabolic conditions. We could create a free-standing bioscaffold by natural drying-mediated assembly from polymercoated liposomes. The membrane of lipo-PArgDNA capsules, which were precipitated over a mesh grid with microholes by using DNA as a dry ligand, possessed both robustness and flexibility. It is expected that such a bioscaffold of lipo-PArg DNA will have capabilities of immobilization of bioactive molecules such as peptides and DNA on the surface and of their efficient transport across the cell membrane by the surfacedisplayed PArg.
ARTICLE
(12) Cheng, W.; Campolongo, M. J.; Tan, S. J.; Luo, D. Nano Today 2009, 4, 482–493. (13) Torchilin, V. P. Pept. Sci. 2008, 90, 604–610. (14) Chugh, A.; Eudes, F.; Shim, Y.-S. IUBMB Life 2010, 62, 183–193. (15) Vives, E.; Richard, J. P.; Rispal, C.; Lebleu, B. Curr. Protein Pept. Sci. 2003, 4, 125–132. (16) Patel, L.; Zaro, J.; Shen, W.-C. Pharm. Res. 2007, 24, 1977–1992. € (17) El-andaloussi, S.; J€arver, P.; Johansson, H. J.; Langel, U. Biochem. J. 2007, 407, 285–292. (18) Brooks, N. A.; Pouniotis, D. S.; Tang, C.-K.; Apostolopoulos, V.; Pietersz, G. A. Biochim. Biophys. Acta, Rev. Cancer 2010, 1805, 25–34. (19) Heitz, F.; Morris, M. C.; Divita, G. Br. J. Pharmacol. 2009, 157, 195–206. (20) Tsogas, I.; Tsiourvas, D.; Nounesis, G.; Paleos, C. M. Langmuir 2005, 21, 5997–6001. (21) Cheng, W.; Campolongo, M. J.; Cha, J. J.; Tan, S. J.; Umbach, C. C.; Muller, D. A.; Luo, D. Nat. Mater. 2009, 8, 519–525. (22) Jakobsen, U.; Simonsen, A. C.; Vogel, S. J. Am. Chem. Soc. 2008, 130, 10462–10463. (23) Licata, N. A.; Tkachenko, A. V. EPL 2008, 84, 20010. (24) Liu, J.; Li, Y.; Li, Y.; Li, J.; Deng, Z. J. Mater. Chem. 2010, 20, 900–906. (25) Glotzer, S. C.; Anderson, J. A. Nat. Mater. 2010, 9, 885–887. (26) Nakajima, N.; Ikada, Y. Bioconjugate Chem. 1995, 6, 123–130. (27) Mertins, O.; Dimova, R. Langmuir 2011, 27, 5506–5515. (28) Conner, S. D.; Schmid, S. L. Nature 2003, 422, 37–44.
’ ASSOCIATED CONTENT
bS
Supporting Information. TEM images of lipo-PArg produced at pH 5.5 and pH 7.4, changes in scattering light intensity of the suspension of polymer-coated liposomes by the addition of Triton X-100, and confocal laser microscopic image of freestanding membranes formed over a mesh SUS grid. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected].
’ ACKNOWLEDGMENT This work was supported by a Grant-in-Aid for Science Research (B) (19300176). ’ REFERENCES (1) Velev, O. D.; Gupta, S. Adv. Mater. 2009, 21, 1897–1905. (2) Rozenberg, B. A.; Tenne, R. Prog. Polym. Sci. 2008, 33, 40–112. (3) Shastri, V. P. Adv. Mater. 2009, 21, 3246–3254. (4) Drury, J. L.; Mooney, D. J. Biomaterials 2003, 24, 4337–4351. (5) Romano, N. H.; Sengupta, D.; Chung, C.; Heilshorn, S. C. Biochim. Biophys. Acta, Gen. Subj. 2011, 1810, 339–349. (6) Heurtault, B.; Saulnier, P.; Pech, B.; Proust, J.-E.; Benoit, J.-P. Biomaterials 2003, 24, 4283–4300. (7) Kulkarni, M.; Greiser, U.; O’Brien, T.; Pandit, A. Trends Biotechnol. 2010, 28, 28–36. (8) Fujimoto, K.; Toyoda, T.; Fukui, Y. Macromolecules 2007, 40, 5122–5128. (9) Fukui, Y.; Fujimoto, K. Langmuir 2009, 25, 10020–10025. (10) Germain, M.; Grube, S.; Carriere, V.; Richard-Foy, H.; Winterhalter, M.; Fournier, D. Adv. Mater. 2006, 18, 2868–2871. (11) Maeda, T.; Fujimoto, K. Colloids Surf., B 2006, 49, 15–21. 9582
dx.doi.org/10.1021/la201500b |Langmuir 2011, 27, 9576–9582
