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Sponge-Like Porous Silica Nanosheets: from “Soft” Molecular Trapping to DNA Delivery Qingmin Ji, Tomohiko Yamazaki, Jiao Sun, #aneta Górecka, Nien-Chi Huang, Shan-hui Hsu, Lok Kumar Shrestha, Jonathan P Hill, and Katsuhiko Ariga ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b15082 • Publication Date (Web): 20 Jan 2017 Downloaded from http://pubs.acs.org on January 22, 2017
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Sponge-Like Porous Silica Nanosheets: from “Soft” Molecular Trapping to DNA Delivery Qingmin Ji,†* Tomohiko Yamazaki,‡* Jiao Sun,† Żaneta Górecka,§ Nien-Chi Huang,ǁ Shan-hui Hsu,ǁ Lok Kumar Shrestha,‡ Jonathan P. Hill,‡ and Katsuhiko Ariga‡ † Herbert Gleiter Institute of Nanoscience, Nanjing University of Science & Technology, 200 Xiaolingwei, Nanjing 210094, China. ‡ WPI Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki, 305-0044, Japan. § Faculty of Materials Science and Engineering, Warsaw University of Technology, Wołoska 141, Warsaw 02-507, Poland. ǁ Institute of Polymer Science and Engineering, National Taiwan University. No.1 Sec. 4 Roosevelt Road, Taipei 10617, Taiwan. ABSTRACT: Sponge-like porous silica nanosheets having nanometer thicknesses and possess pores whose diameters are on the hundreds-of-nanometers scale have been used as a novel carrier for molecular immobilization of different guests. Enhanced properties of encapsulation were shown for drug molecules of different dimensions due to "softness" caused by the specific nanometric features of the porous structure. The encapsulating effect of the structure results in sustained and stimuli-responsive release behavior of immobilized guest molecules. By studying the adsorption process of DNA molecules on the sponge-like porous nanosheets or on solid nanoparticles using a quartz crystal microbalance, we prove that the better elasticity of the surfaces of the porous nanosheets over that of solid nanoparticles can improve the immobilization of guest molecules. The coating of porous silica nanosheets onto various substrates was also found to effectively mediate DNA delivery to mammalian cells. Keywords. porous, nanosheet, silica, control-release, DNA delivery
1. INTRODUCTION Porous substrates, which possess large surface areas and channel spaces, provide suitable platforms for trapping of molecules or nanostructures at their interiors.1-5 This encapsulation or immobilization of different guest atoms, molecules, or nanostructures is essential to ensure the incorporation of the respective functionalities,6-10 and allows the creation of novel systems in optics, electronics, medicine, separations, sensing, and catalysis fields.11-17 The driving force for guest encapsulation in a porous host can be based on covalent bonding or various physical interactions including van der Waals forces, electrostatic interactions or capillary force.18-22 However, controllable encapsulation with effective binding, loading and performance depends largely on the pore structural parameters. Therefore, the exploration of novel porous substrates from the design perspectives of morphological molecular construction, processing, and surface modification is still a challenging area of research. The continually growing demand for efficient drug delivery systems has promoted the development of new porous substrates that can be used to regulate the encapsulation and release of pharmaceuticals, proteins, or genes. Various porous systems based on the networked structures of synthetic polymers, nanoparticles, carbon nanostructures, and supramolecular structures, etc., have been prepared for this purpose.23-31 In particular, silica mesoporous structures have attracted extensive interest due to their biocompatibility, high porosities and simple procedures for modification.32-37 However, the most effective binding and release in silica
mesoporous structures usually involves precise molecular design, which in turn implies complicated synthetic procedures. In addition, silica particles lack morphological flexibility due to their rigid pore structures, where pore dimensions can be adapted for small guest molecules, but cannot always be modified to accommodate large molecules. On the other hand, particles with large pore sizes may lead to
Scheme 1. Scheme for (a) the formation of porous silica nanosheets on a substrate; (b) the porous silica nanosheets peeled from the substrate as a container for trapping molecules; (c) microchanges of nanosheets for the controlled release and delivery.
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unexpected guest release in the absence of any surface modification. With this in mind, a substrate might be more suitable for these purposes if easily accessible spaces of the appropriate dimensions and morphological flexibility can be achieved for guest binding of various molecules. Here, we present a novel silica nanosheet material as a candidate for support substrate or carrier, which is capable of encapsulating various molecular species without any surface modification (Scheme 1). These silica nanosheets are of several nanometers in thickness.38-41 In this work, we peeled the silica nanosheets from their supporting substrate after the fabrication process and used them as colloids for binding of molecular guests. The sponge-like network of silica nanosheets possesses a porous morphology on the several hundred-nanometers microscale, which might behave as an easily accessible container space for adsorbent molecules. Since the one-pot formation process involves large amounts of BO2- ion, different borate linkages may be associated with the framework of silica nanosheets.38 Subsequent removal of borate ions by washing leaves mesopores in the nanosheets and also results in an incomplete siloxane network. In addition, since no heat treatment is applied, a large quantity of silanol groups remain in the structure. These morphological features of the nanosheets (nanometer-scale thin nanosheets, large numbers of surface silanol groups, mesopores, highly networked nanosheets) make the porous silica nanosheets capable of adapting their morphologies when adsorbing molecules, thus providing an effective trap for the adsorbed molecules (e.g. UV-adsorption drug and DNA) at their surfaces, possibly through simple hydrogen bonding and porosity effects. This enhancement of molecular binding leads to a much improved sustained release behavior of the encapsulated molecules whose release rate can only be increased by sonication (several seconds) or partial dissolution of the network support. When compared to other silica porous structures (which are usually subjected to surface modification for binding molecules), these silica nanosheets may avoid problems of toxicity introduced by the surface ligands used in biological applications. In order to illustrate the morphological flexibility of the silica nanosheets for binding molecules, we attempted to study the adsorption kinetics by quartz crystal microbalance (QCM). By observing the adsorption process of DNA molecules on the solid surfaces of the porous silica nanosheets or solid nanoparticles, we prove that more elastic binding occurs between silica nanosheets and DNA. The flexibility of the structures formed from porous silica nanosheets may also be utilized for the controlled release of molecules. Silica nanosheets can also be deposited on the surfaces of various bio-related substrates (slide glass, polystyrenes and steel), and successfully mediate DNA delivery to mammalian cells from those surfaces. The results described in this paper constitute another successful example where a relatively easily prepared material performs as a universal container (solid support) for molecules of various dimensions.
2. EXPERIMENTAL SECTION 2.1 Preparation of silica nanosheets. SiO2 thin film of 1 mm thickness was formed on a single-side polished Si(001) 6 inch diameter wafer by magnetron sputtering deposition. The Si wafer was properly cleaned and dried by spinning before being loaded in the sputtering system. A manufacturing-scale
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apparatus, Shibaura Mechatronics (OCTAVA-II), was used for sputter deposition, along with either an RF (13.56 MHz) or a DC power supply with an arc suppression function. A high purity (99.99%) SiO2 target was used with high purity Ar gas. The silicon wafer containing the 1 mm thick silica layer was cut into 2 cm × 2 cm pieces for convenience. These were then immersed in NaBH4 solution (0.10 g/ml, 2ml) in a 20 mL capacity teflon-lined steel autoclave and incubated at 75 °C for 6 hours. After reaction, the solution was sonicated for 1 minute. Samples contained in the solution were collected by centrifugation and were rinsed with pure water several times until the washings were of neutral pH. A further dialysis process was also carried out in water using Slide-A-Lyzer 2K Dialysis Cassettes (Thermo Sci.) for 1 day duration. The samples were finally freeze-dried under vacuum at room temperature. 2.2 Immobilization of molecules in the silica nanosheets. An ethanol solution of PBSA (0.05g L-1, 2 mL) or 0.01 mg/ml DNA (2 mL) aqueous solution was mixed with dry silica nanosheets (2 mg) or silica nanoparticles (80 nm, Nissan Kagaku Co.) and stirred for 2 h at room temperature. The dispersions were then left to stand at room temperature overnight. The samples were collected by centrifugation and washed with pure water. The loading amount was estimated by measuring the concentration change in the supernatant by UVvis spectrophotometry. The release of PBSA or DNA from the networked nanosheets was carried out by dispersing the mixture (1 mg) in a vessel containing pure water (2 mL) or buffer solution with agitation by shaking at room temperature. A 0.5 mL aliquot was periodically drawn from the medium to follow the release of PBSA or DNA then returned to the same vessel in order to maintain a constant volume. The released amounts of PBSA were monitored by optical absorbance at 300 nm. The released amounts of DNA were monitored by monitoring optical absorbance at 265 nm. 2.3 Encapsulation of drug molecules in the nanocapsules. Recording molecular encapsulation in structures. The binding process of DNA on the surface of the silica nanosheets or silica nanoparticles was recorded using a quartz crystal microbalance (QCM) (Affinix QN pro; Initium, Tokyo, Japan). The QCM instrument has a 500 L cell equipped with a 27MHz QCM plate (AT-cut shear-mode quartz plate and gold electrode with a 4.9 mm2 area) at the bottom of each cell. The unit was equipped with a stirrer bar and a temperature controller. The change in frequency was monitored using a frequency counter attached to the computer software. The frequency shift depends on the adsorbed mass in accordance with the Sauerbrey equation42:
F
2
2 F0 m A q q
In the Sauerbrey equation, ΔF is the measured frequency shift (Hz), Δm is the mass change (g), F0 is the fundamental frequency of the quartz crystal (2.7×106 Hz), A is the electrode area, ρq is the density of quartz (2.65 g cm-3), and μq is the shear modulus of quartz (2.95×1011 dyn cm-2). According to the above equation, at 27 MHz, a frequency shift of 1 Hz corresponds to a mass change of approximately 0.62 ng cm-2.
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The oscillation energy loss (1/Q) related to the viscoelastic property of the surface was estimated based on the following equations:
1/ Q
2( F2 Fs ) Fs
Fs is the series resonance frequency (Hz) and is equivalent to the frequency measured by the conventional oscillation method. F2 is the frequency (Hz) at which the viscous load of solution can be cancelled. In brief, the porous silica nanosheets or silica nanoparticles were immobilized on the surface of a QCM sensor. After adding 100 L DNA solution into the vessel of the QCM sensor, the time course of the frequency changes was monitored. The binding amount of DNA molecules and the energy loss related to the viscous and elastic friction were calculated from curve fitting to the QCM frequency decrease. 2.4 Transfection to mammalian cells on substrates coated with silica nanosheets. Human embryonic kidney cell lines, HEK293XL/null cells (InvivoGen, San Diego, CA, USA) were used for transfection analysis. Cells were cultured in DMEM medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10 vol% FBS, 50 U/mL penicillin, 50 g/mL streptomycin at 37 ºC in a moisture chamber containing 5% CO2 in air. The resuspended solution of silica nanosheets was dropped
and spread on the surface of the slide glass, polystyrene plate or SUS316L steel. In the case of steel, poly(allylamine hydrochloride) (PAH, MW5000, 0.5wt% solution) was precoated by dipping to assist the spreading of the silica nanosheets. Nanosheet-covered substrates were placed in a 24 well plate and then sterilized by applying UV irradiation. phMGFP vector (1 g, Promega, Madison, WI, USA) or pGL4.13 vector (Promega) were diluted with Opti-MEM Medium (50 L, Thermo Fisher Scientific, Waltham, MA, USA) and then Lipofectamine 2000 transfection reagent (50 L, Thermo Fisher Scientific) was added. After incubation for 10 minutes, plasmid DNA/Lipofetcamin 2000 complex (100 L) was added into the 24 well plate containing the substrate and incubated for 30 minutes at room temperature. The substrates were then washed three times each with pure water (1 mL) to remove free plasmid DNA/Lipofectamin complex. HEK293XL/null cell suspension (1 mL, 2.0 × 105 cells/mL) was then added to each well of the tissue culture plate and incubated in 5% CO2 at 37 °C for 48 hours. After another 48 h, the cells were harvested using PLB lysis buffer (Promega), then the luciferase expression level was measured using the luciferase assay system (Promega). The expression of green fluorescence in cells was observed using a fluorescence microscope (DMIL, Leica Microsystems, Wetzlar, Germany).
Figure 1. SEM image of (a) continuous silica nanosheet film grown on the Si substrate (without peeling) and (b), (c) porous silica nanosheets peeling from the substrate; (d), (e) and (f) TEM images of the peeled porous silica nanosheets; (g) EDX spectrum of porous silica nanosheets.
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3. RESULTS AND DISCUSSION 3.1 Porous Morphology from Silica Nanosheets. The silica layer deposited on the Si wafer could be transformed into a porous structure of silica nanosheets by a similar dissolutionregrowth process as previously reported.43-45 As shown in Scheme 1a, during a dissolution-regrowth process, NaBH4 solution provides basic conditions for the dissolution of the silica layer and the decomposition product (BO2 ions) from reaction with water then promotes the polymerization of the dissolved silicate species. With prolonged reaction time, the regrown silica layer transforms into a crosslinked network of nanosheets rather than simply becoming a thicker film.43 The partially vertically regrown nanosheets are not formed directly with the self-templating layer of silica. An aqueous interlayer exists between the original layer and the new grown silica due to the charge repulsive force between dissolved silicate and the bottom silica coating. Therefore, the regrown silica nanosheets can be easily peeled off from the surface of the coating layer by sonication after the reaction and dispersed into water as a colloid solution (Figure S1). It should be mentioned that the collected silica nanosheets were thoroughly washed with pure water to remove salts formed in the formation process. Figure 1b-1d shows SEM and STEM images of regrown silica nanosheets collected from the reaction. The images indicate that the networked morphology of silica nanosheets was maintained, although being somewhat fragmented compared with the continuous film of nanosheets prior to peeling from the surface (Figure 1a). The width of the porous silica nanosheets is 5-20m with voids of several hundred nanometers in size. By observing the edge of the porous layer, its thickness was found to be ~100 nm, while the nanosheets themselves have thicknesses of ~10 nm (Figure 1e). High resolution TEM imaging reveals the mesoporous structure of amorphous silica (Figure 1f). EDX analysis confirms that Si and O are the main composition elements in the silica nanosheets (Figure 1g). We did not find any obvious signals assignable to Na (1.07 keV) in the EDX spectra, which also indicates the complete removal of sodium salts from the nanosheets during the washing process. Since the preparation of these porous silica nanosheets does not require any heat treatment, large amounts of silanol group remain on their surfaces. A comparison of Fourier transform infrared (FTIR) spectra of the porous silica nanosheets with silica nanoparticles of 80 nm in-diameter reveals that silica nanosheets possess more highly active surfaces than silica nanoparticles (Figure S2). The spectrum of silica nanosheets contains stronger adsorption around 1650 cm-1 and 3500 cm-1, which is associated with Si-O-H bending and stretching vibrations.46-48 The band around 1100 cm-1 due to Si-O-Si stretching vibration, also exhibits a red shift in the silica nanosheets compared to silica nanoparticles. This implies that incomplete Si-O-Si linkages exist in the silica nanosheets, which also might lead to the presence of more silanol groups in the silica frameworks. Nitrogen sorption analyses indicate that the porous nanosheets have a Brunauer-Emmett-Teller (BET) specific surface area and a pore volume of 525 m2 g-1 and 0.97 cm3 g-1, respectively. The isotherm of the porous nanosheets is type IV, indicating the presence of mesopores in the nanosheet (Figure S3). These structural features of the sponge-like silica nanosheets should dominate the adsorption and encapsulation activity of molecules in the structure.
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3.2 Adsorption of Molecules in Porous Silica Nanosheets. For drug carrier applications, sufficient space is required in the structure to ensure the loading or encapsulation of the drug molecules. The porous silica nanosheets differ from the silica spheres or mesoporous structures generally used as drug carriers in that, although the loading space mostly originates from the staggering of nanosheets in their network, the highly open accessible surface of nanosheets may favor penetration of molecules into the structure. The mesoporous surface may also facilitate the adsorption of the molecules at the surface. When plasmid DNA, a relative large molecule, was encapsulated in the networked nanosheets, a higher loading efficiency was observed. Based on the concentration change of the supernatant of the DNA-nanosheets mixture solution determined using UV-vis spectroscopy, there was a 34% loading efficiency of DNA inside the networked nanosheets compared with only a 3.3% efficiency for silica nanoparticles with 80 nm in diameter (Figure 2a). Elemental mapping on the DNA loaded silica nanosheets also indicates the successful binding of DNA to the structures (Figure S4). It should be noted that the surfaces of the networked silica nanosheets were
Figure 2. UV-Vis spectra for (a) the original DNA solution (i), supernatant solution obtained by mixing the DNA solution with silica nanoparticles of 80 nm diameter (ii) and porous silica nanosheets (iii); (b) the original PBSA solution (i), supernatant solution from mixing PBSA solution with silica nanoparticles of 80 nm diameter (ii) and porous silica nanosheets (iii). The inset shows the calculated loading efficacies of silica nanoparticles and porous silica nanosheets.
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not modified to be positive so that there is no electrostatic force involved in the loading process. Zeta potential measurements indicate that the surface charge of the silica nanosheets is -23 mV and changes to -29 mV after loading with DNA. Considering the existence of large silanols in the structures, the hydrogen bonding between silica nanosheets and DNA may play roles for the binding. The porous structure of the silica nanosheets itself may also promote a more effective adsorption of DNA. From the FTIR spectrum of DNA loaded silica nanosheets, we can find that there is obvious change at the Si-O-Si stretching vibration, which indicating the adsorption of DNA on the framework of silica. The broad adsorption from 1500-1720 cm-1 also implied the interaction between silica and DNAs (Figure S5). The DNAs loaded in silica nanosheets are stable with time (5 days) based on the UV-vis spectra. We not observed obvious shift at adsorption peak of DNA (265 nm) in the dispersion of DNA loaded silica nanosheets (Figure S6). According to STEM and SEM images of the nanosheets loaded with DNA (Figure 3a and 3b), it is apparent that there are some variations in the wave-like form of the nanosheets. The average thickness of the nanosheets had also increased from 10 nm to 30 nm after the adsorption of DNA (Figure 3b). This morphological variation by adsorption may facilitate the immobilization of DNA in the structure. We also studied the adsorption on the nanosheets of the small drug molecule, 2-phenylbenzimidazole-5-sulfonic acid (PBSA), a therapeutically important UV-absorber,49-51 An ethanolic solution of PBSA, was mixed with the porous silica nanosheets. The loading capacity of the nanosheets (15%) is nearly 10 times greater than that of silica nanoparticles of 80 nm in diameter (3%) (Figure 2b). The porous silica nanosheets also appeared to have a higher morphological density after loading of PBSA (Figure 3c and 3d). These results indicate that the porous structures of silica
Figure 3. (a) STEM and (b) SEM images of porous silica nanosheets after loading DNA. (c) STEM and (d) SEM images of porous silica nanosheets after loading PBSA.
nanosheets have enhanced adsorption capabilities, which do not depend on molecular dimensions of the adsorbents, and also do not require any surface modification of the nanosheets for optimization of the interactions between molecules and the substrate. These “soft” thin nanosheets and the mesopores in the structure may lead to a relatively flexible surface (compared with other rigid substrates). These observations might be due to the micro-deformation of the porous silica nanosheets caused by the adsorption leading to a partial loss of the kinetic energy of the surface-adsorbed molecules, which in turn reduces the motion of the molecules within the structure.
Figure 4. Dynamic adsorption process of (a), (c) porous silica nanosheets and (b), (d) silica nanoparticles encapsulating DNA molecules in water. (a), (b) Frequency changes and (c), (d) viscoelastic loss energy based on QCM recording upon immersion in water and addition of DNA.
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Larger friction forces between the surface of silica nanosheets and molecules may also enhance the immobilization of molecules. To study the effect of nanosheet morphological features on the adsorption of molecules, we compared the dynamic adsorption behavior of DNA molecules on the porous silica nanosheets and of silica nanoparticles by using a quartz crystal microbalance (QCM). The QCM measurement can not only record the real-time absorption by frequency shift, but also the surface viscoelastical changes during the loading process. The amount of the silica nanosheets or silica nanoparticles loaded onto the surface of the QCM sensor was estimated according to the frequency changes (ΔF) before and after coating. We adjusted the coating amount so that ΔF ~2000 Hz, which is equivalent to about 1.2 g cm-2. Prior to the adsorption of DNA molecules, the QCM sensors containing silica samples at their surfaces were stabilized in aqueous solution. On addition of DNA to the solution, the frequency decreased almost immediately indicating the adsorption of DNA on the surface. Representative plots of the QCM results for the adsorption of DNA on the substrates are shown in Figure 4. ΔF varies by around 200 Hz for silica nanosheets, while only a 30 Hz change occurs for the silica nanoparticles, corresponding to adsorption quantities of 124 and 18.6 ng cm-2, respectively. It should be noted that the loading efficacies found by QCM are much lower than those obtained by UV-Vis spectrophotometry. This is because the “wrapping” ability of the networked silica nanosheets is substantially reduced due to adsorption on a supporting substrate rather than being unrestrained in solution. We also found differences in adsorption by silica nanosheets and silica nanoparticles when considering their adsorption plots (Figure 4a and 4b). DNA adsorption on the surface of silica nanoparticles quickly reached saturation (100s). Conversely, in the case of silica nanosheets, the adsorption behavior of DNA varied gradually following an initial sudden decrease in frequency, and reached a saturation point over a relatively long 600 s time period. These results imply that the network of silica nanosheets has greater vacant space for the loading of guests, and also that the channels at their interiors are interconnected permitting the penetration of the molecules into the network. Through an analysis of viscoelastical properties based on the frequency shifts from the energy loss, we can further define the surface conditions prevailing during adsorption. The oscillation energy loss (1/Q) is related to the viscoelastical changes from the elastic region of the solid and viscous part of the solution. When immersed in water, 1/Q exhibits an immediate increase with a gradual return to the starting state (Figure 4c). This change describes the process of water contacting the substrate to its spreading on the surface. 1/Q changes much more drastically for silica nanosheets over silica nanoparticles. This observation is likely due to the larger space contained at the interior of the nanosheets, which causes a rapid suction effect of water, and the penetration of water into the nanosheets resulting in the formation of a more gel-like structure. The soft surface of the porous silica nanosheets in water leads to a less elastic response during the subsequent adsorption of DNA than during the same process in silica nanoparticles (Figure 4c and 4d). This suggests that the silica nanosheets provide a similar elastic surface to DNA molecules. The gel-like surface of silica nanosheets is substantially dependent on their sponge-like porous states. To further understand the adsorption behavior on the solid
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substrate, we used Flory-Huggins adsorption model to fit variations occurring during the adsorption process of DNA on the surfaces of silica nanoparticles or silica nanosheets. As shown in Figure S7, the data obtained for the adsorption in each case can be well plotted as a linear relationship based on the equation of Flory-Huggins isotherm. According to the linearized equations, the equilibrium constants KFH for silica nanosheets and silica nanoparticles are 3.47 and 2.27, respectively. KFH can be used for the calculation of free Gibbs energy for the adsorption behavior. Thus, values obtained for ΔG0 for the adsorption of DNA on silica nanosheets and silica nanoparticles are -20 kJ mol-1 and -12 kJ mol-1, respectively. ΔG0 expresses the feasibility and spontaneity of the adsorption process. A relatively low Gibbs energy for the case of silica nanosheets implies a more spontaneous adsorption of DNA on the surfaces of silica nanosheets than for silica nanoparticles. On the other hand, perhaps due to the incomplete siloxane framework of the silica nanosheets (and also the voids left by salts during the formation process), the structure was found to be variable in buffer solution (Figure S8). When immersed in PBS buffer solution (pH 7.4), the sponge-like structures with pores at their interiors are likely gradually transformed into thicker membranes with a wave-like form with a consequent significant decrease in the void space of the network. BET specific surface area and pore volume of the porous silica nanosheets after immersion in PBS buffer for 10 hours
Figure 5. Cumulative release profiles of DNA from porous silica nanosheets while varying the medium to PBS buffer solution, and (b) Cumulative release profile of PBSA from (a) porous silica nanosheets under short intervals of sonication.
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decreases by 40% (314m2 g-1) and 30% (0.71cm3 g-1), respectively (Figure S9). We also used inductively coupled plasma-atomic emission spectrometry (ICP-AES) to detect the changes of silica nanosheets when immersed in PBS buffer solution. With longer immersion time (after 7 days), ICP-AES of Si in the supernatant gradually increased until almost 90% Si was dissolved in the PBS buffer solution (Figure S10). In comparison, almost no Si can be detected in the supernatants from the dispersions of DNA loaded silica nanosheets (Figure S10) at different storage times. We suggest that the intercalation of salt ions in the buffer solution may disrupt the silica framework leading to morphological variation of the structure. To study this morphological effect on the binding of DNA, we compared the adsorption behavior of the porous silica nanosheets with and without DNA loading under the PBS buffer solution by QCM (Figure S11). The results indicate that the mass of the porous silica nanosheets slightly decreased in the buffer solution, which should be due to the partial dissolution of Si from the surface by the intercalation of ions in the structures. Concurrently, the 1/Q value was shown gradually to decrease due to a reduction in viscoelasticity, in turn implying that the porous structure changes from "soft" to "hard". In the case of the porous silica nanosheets containing DNA, the mass (ΔF) obviously decreased following immersion in the PBS solution for several hours (Figure S11), indicating that DNA in the structure was released out due to the morphological changes. Thus, the variation of the network
morphology triggers the adsorption of molecules in the porous structure of silica nanosheets. 3.3 Stimulus-Release Behavior of Molecules. Despite their open and accessible porous structures, the guest molecules loaded in the silica nanosheets did not exhibit rapid release as was the case for silica nanoparticles (Figure S12). On the contrary, a sustained release behavior was shown for the encapsulated molecules. Just ~30% of DNA contained in the porous nanosheets was released within 15 hours (Figure 5a). For encapsulated PBSA, after an initial rapid release (35%) during the first 5 min, release became relatively slow in the subsequent 2 hours (Figure 5b). We believe that this contrasting behavior is due to the above mentioned morphological changes of the nanosheets when loaded with guest molecules. There may be a partial covering or "wrapping" effect of the molecules encapsulated inside the porous structures. Our attempts to prove the operation of this effect involved "unwrapping" of the network by applying some external stimulus. Considering the ease with which DNA can be denaturated, we chose a mild stimulus to the vary the morphology of silica nanosheets. We thus applied the PBS buffer solution to the DNA loaded silica nanosheets, which can break the network structure without influencing the structure and activity of DNA. In the release profile of DNA, it can be observed that the release rate obviously increases in PBS buffer solution compared to that in pure water. The re-
Figure 6. (a) Scheme of Gene transfection to 293xl/null cells mediated by suspended silica nanosheets deposited on substrates. Fluorescent images of 293xl/null cells, which were transfected with GFP coded plasmid on (b) a bare glass surface and (c) on slide glass deposited with silica nanosheets on the surface. Quantative analysis of gene transfection by (d) slide glass, (e) polystyrene (PS), and (f) steel (SUS316L) with and without silica nanosheets by measuring luciferase activity. Data are averages of three parallel experiments. Statistical significance was calculated using a two tailed unpaired Student’s t-test. * p-value of
