Study on the Adsorption Mechanism of DNA with Mesoporous Silica

Nano Biomedical Research Center, School of Biomedical Engineering and Med-X Research Institute, Shanghai Jiao Tong University, 1954 Huashan Road, ...
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Study on the Adsorption Mechanism of DNA with Mesoporous Silica Nanoparticles in Aqueous Solution Xu Li, Jixi Zhang, and Hongchen Gu* Nano Biomedical Research Center, School of Biomedical Engineering and Med-X Research Institute, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai 200030, China S Supporting Information *

ABSTRACT: Among the numerous adsorption strategies for DNA adsorption into mesopores, the salt-solution-induced adsorption method has a great application potential in nucleic acids science; thus, it is important to understand the adsorption mechanism. This work demonstrates the mechanistic aspects underlying the adsorption behaviors of DNA with mesoporous silica nanoparticles (MSNs) in aqueous solution. The driving forces for the adsorption process can be categorized into three parts: the shielded electrostatic force, the dehydration effect, and the intermolecular hydrogen bonds. Compared to the adsorption behaviors of DNA with a solid silica nanosphere, we find some unique features for DNA adsorption into the mesopores, such as increasing the salt concentration or decreasing the pH value can promote DNA adsorption into the mesoporous silica. Further analysis indicates that the entrance of DNA into mesopores is probably controlled by the Debye length in solution and DNA can generate direct and indirect hydrogen bonds in the pores with different diameters. The following desorption study depicts that such types of hydrogen bonds result in different energy barriers for the desorption process. In summary, our study depicts the mechanism of DNA adsorption within mesopores in aqueous solution and sets the stage for formulating MSNs as carriers of nucleic acids. within MSN mesopores. Solberg and Landry22 utilized multivalent cations such as Mg2+ and Ca2+ to mediate the packaging of DNA into MSN mesopores. Gao and colleagues23 modified the internal surface of MSN mesopores with cationic linkers and then used such functionalized MSNs adsorbed plasmid DNA molecules. In these cases, although the electrostatic force successfully induced DNA adsorption into MSNs, the detachment of DNA, an essential requisite for DNA extraction or manipulation,24,25 was blocked by the strong interaction between the sorbent and absorbent. Consequently, other researchers tried to avoid making use of electrostatic interaction to achieve the loading of DNA within MSNs. Fujiwara and co-workers26 found DNA could be adsorbed into MSN mesopores in aqueous solution with high NaCl concentration and low pH values. Our groups27 utilized the chaotropic salt solution (2 M Guanidine hydrochloride, pH ∼5) to mediate short salmon DNA (∼150 bps) adsorption into a type of magnetic mesoporous silica nanoparticles (M-MSNs). Through comparing the DNA adsorption and desorption behaviors between M-MSNs and a type of solid silica nanoparticles (SNs) with similar particle size, we have proven that abundant DNA molecules were unequivocally encapsulated within M-MSN mesopores. These studies on the saltsolution-mediated DNA adsorption suggested that the mesoporous silica could serve as good carriers for DNA

1. INTRODUCTION Since the discovery of surfactant-templated synthesis of mesoporous silica materials in 1992,1 considerable effort has been devoted to exploring the functionalization and utilization of these materials for different applications, such as catalysis,2−4 separation,5,6 and sensors.7,8 Nevertheless, because of the lack of morphology control, there was no report about the application of these materials in the biomedical field for drug delivery and controlled release until the mesoporous silica nanoparticles (MSNs) appeared in the early 2000s.9,10 Compared to the irregularly shaped bulky mesoporous materials, MSNs have uniform and tunable particle size typically on the nanolevel, which is fundamentally important for mammalian cells engulfment and in vivo systematic application within living bodies.11,14 The retaining features of mesoporous materials such as high surface area and pore volume may allow well controllable loading and release of guest molecules.9,11 Consequently, many attentions have been drawn by developing the possibilities of MSNs as carriers for small drugs,12−14 proteins,15−17 enzymes,18−20 and so on. Since the great success of applying MSNs as drug delivery systems, recently, researchers attempt to utilize this material for the packaging of duplex DNA molecules. Nevertheless, DNA is a type of bulky biomolecule with high negative charge density on its backbone,21 thus the entrance of DNA into MSN mesopores with a negatively charged silica surface becomes very hard in aqueous solution conditions. Up to now, only a small amount of studies has been reported for DNA adsorption © 2011 American Chemical Society

Received: November 11, 2011 Published: December 19, 2011 2827

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Figure 1. Ascending adsorption isotherms (25 °C) for salmon DNA with solid silica nanoparticles without pores (A) and M-MSNs (B), respectively, in 2 M guanidine-HCl with pH 5.2. A Langmuir fit was used for each group of data to estimate the maximum adsorption capacity. 2.3. DNA Adsorption into M-MSNs. An adsorption isotherm was obtained by preparing a series of salmon DNA solutions with initial concentrations ranging from 50 to 2000 μg/mL in deionized water. In each experiment, 10 μL of the DNA solution was added into a 0.5 mL centrifuge tube with 0.2 mg of M-MSN and 10 μL of deionized water prepared before, and then, 20 μL of binding buffer (4 M GuanidineHCl, pH ∼5) was added. The mixture was well-dispersed by vortex for 30 s and then continuously shook at 25 °C for 20 h. The final solution was placed under magnetic field for 2 min to separate the M-MSN from the supernatant liquid. The amount of DNA adsorbed by MMSNs was calculated from the depletion of DNA concentrations in solutions after adsorption. To study the DNA adsorption capacities of M-MSNs influenced by the varying binding solutions, the amounts of adsorbed DNA by MMSNs were determined under different adsorption conditions, including NaCl solution with salt concentrations changing from 0.1 to 2.5 M (with a fixed pH ∼5.2), guanidine-HCl solution with salt concentrations changing from 0.1 to 2.5 M (with a fixed pH ∼5.2), and guanidine-HCl solution with pH values changing from ∼4 to ∼8 (with a fixed salt concentration of 2 M). 2.4. DNA Desorption from M-MSNs. All the M-MSN samples were saturatedly adsorbed with DNA before desorption study throughout this work. After adsorption for 20 h, the M-MSN sample was separated and washed with ethanol twice to ensure that residual chaotropic salts were removed. Then, the M-MSN sample was directly suspended in 40 μL of elution buffer (150 mM NaCl) in a 0.5 mL centrifuge tube and incubated at 0.5 °C immediately. After incubation at 0.5 °C for 1 h, the temperature was raised up to 5 °C within 2 min, and the DNA concentration in elution buffer was measured. This process was repeated by increasing the temperature by 5 °C in each round until the final desorption temperature was raised to 40 °C. For all the adsorption and desorption measurements, duplicate samples were run, and the final result was the average of such two samples. 2.5. DNA Conformation in Binding Solution. The circular dichroism (CD) spectra data for DNA in 2 M guanidine-HCl was recorded at room temperature by using a JASCO J-815 spectrometer. The DNA concentration was about ∼90 μg/mL. 2.6. Heat Change in Adsorption Process. The adsorption heat change during the adsorption process of DNA with M-MSNs was measured in an isothermal microcalorimeter MicroCal iTC200 (GE Healthcare, USA). Before titration, 10 mg of M-MSNs and 200 μL of binding solution (2 M guanidine-HCl) were mixed in the ampule and stirred at 120 rpm. When thermal equilibrium between the ampule and the heat sink was reached, 40 μL of binding solution with 10 mg of dissolved DNA was titrated into the dispersed M-MSN suspension with a Hamilton syringe within 60 min.

molecules. Based on this success, we also achieved encapsulating small interference RNA (siRNA) within the mesoporous structure of silica.28 Consequently, it is very important and necessary to understand the mechanism of DNA/siRNA adsorption within the mesopores, which is the foundation of setting new approaches for the delivery of nucleic acids and thus actualizing clinical gene therapy. Herein, we devoted our efforts to making clear such an adsorption mechanism of DNA within the mesoporous structure of silica in aqueous solution. We investigated the DNA loading capacities of mesoporous and solid silica nanoparticles, respectively, with changing the solution conditions including the pH values, the salt concentrations, and the types of salts. On the basis of comparing those experimental data, the driving forces for DNA adsorption into the mesoporous structure were analyzed. The DNA conformational transition in chaotropic solution and the heat change during the adsorption process were also performed to interpret the adsorption mechanism. Thereafter, we investigated the correlation between the DNA desorption behaviors and the mesopore diameters, thus indicated the different statuses of intermolecular hydrogen bonds for DNA existing in the mesopores.

2. EXPERIMENTAL SECTION 2.1. Materials. The silica materials used in this work were the same as those utilized in our previous work.27 In brief, the mesoporous silica nanoparticles with core−shell structure were synthesized via the work reported by Hyeon’s group29 and denoted as M-MSNs. Such a MMSN has a magnetic core embedded in the mesoporous silica shell with the particle size of ∼50 nm and the pore size ∼2.7 nm (Figure S3 in Supporting Information). The solid silica nanoparticles with similar particle diameter were synthesized through the Stober method and denoted as SNs as the contrast particles for comparing to the M-MSN. Double-stranded salmon DNA was purchased from Sigma and used as supplied. As stated by the manufacturer, the DNA was cleaved into short segments by sonication and extracted in saturated NaCl solution. According to our previous work,27 the salmon DNA had a distribution from 20 to 250 bp with an average chain length of ∼50 nm. Guanidine hydrochloride (Guanidine-HCl) and NaCl were purchased from the Sinopharm Chemical Reagent Co., Ltd., China. These salts were dissolved by deionized water and prepared as binding buffers with different concentrations. The pH values for those binding buffers were adjusted to fixed values by adding 1 M NaOH or HCl. 2.2. DNA Concentration Measurement. The DNA concentration in solution was determined by using a NANODROP 1000 spectrophotometer (Thermo Scientific, USA), and the final result of concentration was an average of duplicate measurements and was calibrated by the standardization plot method that has been reported in our previous work.30

3. RESULTS Figure 1 showed the ascending adsorption isotherms for DNA with solid silica nanoparticles (SNs) without pores (A) and MMSNs (B), respectively. The two groups of data fitted well with the Langmuir adsorption model, and the maximum adsorption capacities of SNs and M-MSNs were 57.8 and 122.2 mg/g, 2828

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Figure 2. Amount of adsorbed DNA into M-MSNs changed as a function of (A) NaCl concentrations and (B) guanidine-HCl concentrations, respectively, with pH 5.2 at 25 °C. (C) The amount of adsorbed DNA into M-MSNs in 2 M guanidine-HCl changed as a function of varying pH values at 25 °C.

Figure 3. Amount of adsorbed DNA on SNs without mesopores changed as a function of (A) NaCl concentrations and (B) guanidine-HCl concentrations, respectively, with a fixed pH 5.2 at 25 °C. (C) The amount of adsorbed DNA on SNs without pores in 2 M guanidine-HCl changed as a function of varying pH values at 25 °C.

respectively. Based on the adsorption isotherms, the equilibrium concentrations of DNA in further experiments were maintained over 250 μg/mL, to ensure all the samples reaching saturated adsorption. To investigate the driving forces for DNA adsorption into MMSNs, we processed the adsorption experiments under various solution conditions. Figure 2A showed the amount of adsorbed DNA into M-MSNs changing as a function of NaCl concentrations. When the NaCl concentration increased from 0.1 to 0.25 M, the amount of adsorbed DNA was nearly zero. Nevertheless, when the NaCl concentration increased from 0.25 to 2.5 M, the amount of adsorbed DNA grew up steadily with the final amount was calculated to about 84.2 mg/g. Figure 2B showed the amount of adsorbed DNA into M-MSNs changing as a function of guanidine-HCl concentrations. The amount of adsorbed DNA grew up steadily companying with the increase of guanidine-HCl concentration. Figure 2C demonstrated the amount of adsorbed DNA by M-MSNs varying with the pH values in binding solutions. A sharp increase of the amount of adsorbed DNA appeared from pH 8.1 to 6.8. Nevertheless, when the pH changed from 6.8 to 4.1, the amount of adsorbed DNA into M-MSNs increased moderately from 75.4 mg/g to 111.5 mg/g. In parallel to M-MSNs, SNs with similar particle diameter were utilized to process DNA adsorption experiments under the same adsorption conditions. Figure 3A showed the DNA adsorption onto SNs influenced by varying NaCl concentrations. When the NaCl concentration increased from 0.1 to 1.5 M, the amount of adsorbed DNA kept nearly negligible. Nevertheless, when the NaCl concentration was further improved to 2 M, the amount of adsorbed DNA got to ∼35

mg/g. Figure 3B demonstrated the amount of adsorbed DNA on SNs changing as a function of varying guanidine-HCl concentrations. When the concentration of guanidine-HCl was below 0.25 M, the amount of adsorbed DNA kept nearly zero. Nevertheless, when the concentration of guanidine-HCl grew up from 0.25 to 0.5 M, the amount of adsorbed DNA increased sharply. Thereafter, the amount of adsorbed DNA increased slightly from ∼40 to ∼50 mg/g, when the concentrations of guanidine-HCl were higher than 0.5 M. Figure 3C showed the amount of adsorbed DNA on silica nanoparticles (SNs) without pores in 2 M guanidine-HCl changing as a function of pH values at 25 °C. When pH value decreased from 8.2 to 6.8, the amount of adsorbed DNA for SNs increased sharply. Nevertheless, when the pH value further decreased, the change of adsorbed amount became negligible. To investigate the DNA conformational change in salt solution, we analyzed the CD spectra of the DNA in aqueous solutions with 10 mM NaCl and in 2 M guanidine-HCl, respectively. As shown in Figure 4, for the sample of DNA in 10 mM NaCl solution, the positive peak appeared at 275 nm, and the negative peak existed at 245 nm, respectively. This result indicated a typical B conformation for DNA under this condition.21 Compared to this, the wavelength of strongest peaks for the DNA in 2 M guanidine-HCl were remarkably similar. Lower magnitude values were recorded at both the positive bands at 220 and 275 nm, which suggested that a minority of DNA double helix has taken a conformational transition from the B to the C form,31 whereas the others still remained as the B conformation. To obtain the heat change during the adsorption process, isothermal titration calorimetry (ITC), a physical technique 2829

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amounts of desorbed DNA were calculated from each group of data by using a “Doseresp” model (in the database of OriginLab Company), and the normalized curves of corresponding DNA desorption ratios were plotted in Figure 6B. Figure 7A showed the accumulated amount of the desorbed DNA in elution buffer at different temperatures for the DNAloaded M-MSN (average pore diameter of 2.7 nm) samples prepared under the binding solutions with different pH values (the adsorption processes were carried out under pHs 4.1, 5.2, and 6.8, respectively). Although the final eluted amounts were different, the changing trends for these samples were similar. We still used the Doseresp model to fit the three groups of data and obtained the maximum amount of eluted DNA. Figure 7B described the normalized curves of corresponding DNA desorption ratios for the three groups of M-MSN samples. The three curves were nearly overlapped, which suggested that the existent status of the DNA in these samples were very similar.

Figure 4. Comparison of the circular dichroism (CD) spectra of salmon DNA existed in aqueous solutions with 10 mM NaCl (pH 7) and in 2 M gGuanidine-HCl (pH 5), respectively. The DNA concentration was ∼90 μg/mL.

used to determine the thermodynamic parameters of interactions in solution,32 was carried out to study our chaotropic-salt-induced DNA adsorption. Figure 5A showed the typical curves obtained in the ITC experiments for DNA titration into blank binding solution, and the positive peaks represented that the ITC response in such a titration process was endothermic. Figure 5B showed the titration data for DNA injected into binding solution with M-MSNs. In the early 30 min, the negative peaks represented that the process was exothermic. After subtracting the dilution effects, the integrated heat change (enthalpy change) due to each injection of DNA was plotted in Figure 5C. In our investigation of DNA desorption behaviors influenced by the M-MSN pore diameters, besides the M-MSN with average pore diameter of 2.7 nm used throughout this work, we utilized another two types of M-MSN samples with different average pore diameters of 3.4 and 4.9 nm, respectively (Figure S3 of the Supporting Information, more details in our previous work33). All three samples were saturatedly adsorbed with DNA before processing desorption study (the adsorption isotherms for the three samples were plotted in our previous work33). Figure 6A demonstrated the accumulated DNA amount in elution buffer at different temperatures for the DNA released out from M-MSNs with different pore diameters. For the three samples, the amount of desorbed DNA at 0.5 °C increased when the pore diameters of M-MSNs were enlarged. As the temperature increased, the amounts of desorbed DNA for all three samples were also increased. The maximum

4. DISCUSSION 4.1. Driving Forces for DNA Adsorption into Mesoporous Silica. In our experiments to plot the DNA adsorption isotherms for M-MSNs and SNs, the two groups of adsorption data produced typical Langmuir isotherms, suggesting the adsorption processes for both DNA with M-MSNs and SNs were monolayer adsorption occurring at the interface between DNA and silica (DNA did not deposit on other already adsorbed DNA molecules). Consequently, the adsorption of DNA with such two types of particles is mainly attributed to the interaction of DNA with the silica surface. Although the adsorption mechanism of DNA with mesoporous silica is still unclear, the mechanism of DNA interaction with the surface of solid silica has already been investigated by Melzak and coworkers in 1996.34 Based on their research, the driving forces for DNA interacting with a silica surface can be categorized into three parts: the electrostatic screening effect, the dehydration effect, and the generation of intermolecular hydrogen bonds. All the above analysis provides us with clues for studying the mechanism of DNA adsorption with M-MSNs in aqueous solution. It is highly possible that the driving forces for DNA adsorption with M-MSNs is the same as that in the adsorption process of DNA with SNs. To prove this hypothesis, we further analyze the experimental data of DNA adsorption with MMSNs and SNs. Since there is a high density of negative charge on both DNA and silica surfaces, the adjacency of sorbent and absorbent, a

Figure 5. ITC data recorded for the interaction of salmon DNA with M-MSNs. (A) The ITC response for the blank titration with DNA; (B) the raw ITC data for the titration of salmon DNA with M-MSNs; (C) the integrated heat of reaction at each injection of salmon DNA. 2830

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Figure 6. (A) Accumulated DNA amount in elution buffer at different temperatures for the DNA released out from M-MSNs with different pore diameters: 2.7 nm (△); 3.4 nm (■); 4.9 nm (○). For all three groups of data, a Doseresp model (in database of OriginLab Company) was used. (B) The normalized curves for the three M-MSN samples at different temperatures. For each curve, the maximum amount of desorbed DNA was obtained from the Doseresp model.

Figure 7. (A) Accumulated desorbed DNA in elution buffer at different temperatures for DNA released out from M-MSN mesopores. The adsorption processes for the three samples were carried out under various pH values. A Doseresp model (in database of OriginLab Company) was used to fit each group of data. (B) The normalized curves of DNA desorption ratios for the three samples at different temperatures. For each curve, the maximum amount of desorbed DNA was obtained from the Doseresp model.

condition. In the work of Melzak and colleagues,34 the adsorption reaction occurring on the interface between DNA and silica can be written as the following equation: DNA (hydrated) + silica (hydrated) + conterions ⇌ neutral DNA− silica + water. Accordingly, decreasing the amount of free water molecules in solution through improving the concentration of guanidine-HCl drives the reaction to the right and thus enhances the adsorption capacity of the absorbent. This agrees well with the result that the amount of adsorbed DNA grows up steadily when the guanidine-HCl concentration increases from 0.5 to 2.5 M (Figure 3B). In addition, we also observe an interesting phenomenon that the critical concentration for DNA adsorption in guanidine-HCl is much lower than that in NaCl solution (Figure 2 and Figure 3), which indicates an enhanced electrostatic screening effect in guanidine-HCl solutions. This is also proven by the results of the zeta potential of M-MSNs measured in both NaCl and guanidineHCl solutions (Supporting Information, Figure S1). This phenomenon cannot be attributed to the dehydration effect, since the amount of chaotropic salt at low concentration (0.1 M for M-MSNs and 0.5 M for SNs) is not sufficient to induce a considerable loss of free water molecules in aqueous solution. On the other hand, based on the theory of the “hofmeister series”, the adsorption of guanidinium cations on the protein surface is much easier than that of Na+ and thus leads to the “salting in” effect on protein at a low-salt concentration.43,44 Consequently, such an enhanced electrostatic screening effect in guanidine-HCl solution may probably be ascribed to the

prerequisite for the generation of hydrogen bonds, is impossible in water solution conditions. Consequently, shielding the electrostatic repulsion force becomes the key for DNA getting close to the silica surface. Since the electrostatic screening effect is determined by the ionic strength in aqueous solution, a high concentration of salt is necessary to decrease the charge screening length (Debye length) and thus shields the negative charges on DNA and the silica surface. When the salt concentration reaches a critical value, the sorbent and absorbent get close enough to generate the intermolecular hydrogen bonds and achieve adsorption. This matches well with our results obtained from the adsorption experiments for M-MSNs and SNs under the same NaCl solutions (Figure 2A and 3A). The critical salt concentrations are 0.5 M for M-MSNs and 2 M for SNs, respectively. In this regard, the electrostatic screening effect should be one of the driving forces for DNA adsorption with M-MSNs. To testify the influence on DNA adsorption by the dehydration effect, guanidine-HCl, a type of chaotropic salt was used to replace NaCl to carry out the same adsorption experiments. The chaotropic salts can intensively capture free water molecules and thus decrease the amount of bound water on the sorbent and absorbent in aqueous solution. Considering the reality that part of the DNA double helix exists as the C conformation (Figure 4) in 2 M guanidine-HCl and the C conformation usually appears at lower humidity when compared with the B form,31 we confirm that the guanidineHCl causes the loss of bound water from DNA molecules, that is, results in the dehydration of DNA in this binding solution 2831

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Figure 8. (A) Route for DNA adsorption into M-MSNs: the double helix bars represented DNA molecules, the blue spheres represented M-MSNs, the black sphere represented the magnetic core, and the white cylinders represented the mesopores. The sketches of the enlarged cross section for (B) empty mesopore, (C) DNA in mesopore with small pore diameter, and (D) DNA in mesopore with large pore size, respectively.

sequently, the ITC experiment offers the direct evidence for the formation of intermolecular hydrogen bonds in the process of DNA adsorption with M-MSNs. This indicated that the generation of intermolecular hydrogen bonds is one of the driving forces. To sum up, in this monolayer adsorption process of DNA with M-MSNs, the electrostatic screening effect decreases the repulsion force between DNA and silica thus making the sorbent get close to the absorbent; the generation of intermolecular hydrogen bonds achieves the interaction of DNA with silica surface; and the dehydration effect strengthens the adsorption through decreasing the amount of free water molecules. In addition, some specific cations such as guanidinium can enhance the electrostatic screening effect in aqueous solution. The above three driving forces for DNA interaction with SNs also dominated the process of DNA adsorption with M-MSNs. Nevertheless, considering the special mesoporous structure in M-MSNs, we further compared and analyzed the adsorption behaviors of DNA with M-MSNs (Figure 2) and SNs (Figure 3). 4.2. Screening Effect and Hydrogen Bonds in Mesopores. Through study of the adsorption behaviors of DNA with SNs in salt solutions (Figure 3A), we found that the DNA adsorption for SNs occurred at a certain NaCl concentration of 2 M and the adsorbed amount kept the same even the NaCl concentration further increased. Compared to this, the amount of DNA adsorbed by MMSNs increased steadily when the NaCl concentration was raised. These results indicated that the adsorption of DNA with M-MSNs was more dependent on the salt concentrations thus suggesting the screening effect for DNA adsorption with such two types of particles was different. For DNA adsorption onto SNs (Supporting Information, Figure S2A), the negative charge on silica may form an electrostatic field surrounding the particle surface, whose thickness is determined by the Debye length in solution

association of guanidinium cations with the sorbent or absorbent. For DNA adsorption with silica, the intermolecular hydrogen bonds usually generate between the phosphate groups on the DNA backbone and the silanol groups on the silica surface. Through comparing the DNA adsorption behaviors under fixed pH values of 4.8 and 8, Melzak and colleagues found that the maximum DNA adsorption capacity was determined by the solution pH values.34 Similarly, we also found an intensive increase of adsorption capacity of M-MSNs (as well as SNs) when the pH decreased from 8 to a lower pH range (Figure 2C and 3C). Usually, the decrease of pH value promotes the protonation of both of the phosphate groups of DNA and the silanol groups of silica.34 Nevertheless, the isoelectric point of DNA is about 4−5,21 which is far away from this pH range. On the other hand, for the geminal silanol groups on the silica surface,35,36 the pKa for the protonation of the second silanol group is about 8.37 Thus, the sharp increase of DNA adsorption from 8 to lower pH should be attributed to the protonation of germinal silanol groups, which intensively increases the binding sites for DNA interaction with the silica surface. To further prove the generation of hydrogen bonds, we processed isothermal titration calorimetry (ITC) to investigate the heat change in the adsorption process. Two tubes filled with binding buffer solution (2 M guanidine-HCl) were prepared to dissolve salmon DNA and M-MSNs, respectively, ensuring the dehydration processes of the sorbent and absorbent occur independently. Thereafter, the DNA solution was titrated into the solution with M-MSNs to obtain the ITC response for DNA interaction with M-MSNs. The result indicates that the adsorption process is exothermic (Figure 5). Thermodynamically, for sorbent and absorbent, dehydration effect, structural rearrangement, and electrostatic repulsion are considered to result in endothermic processes,38−40 but chemical reactions, electrostatic attraction, and intermolecular hydrogen bonds usually lead to exothermic courses41,42 in a system. Con2832

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diameter of 4.9 nm always gets higher desorption percentages than the other two samples with smaller pore sizes. In addition, as mentioned above, the water molecules can participate in the generation of indirect hydrogen bonds. For DNA adsorption at a low NaCl concentration (such as 0.5 M), the indirect hydrogen bonds cannot fix DNA on the SN surface through a side-on manner (negligible adsorbed amount in Figure 3A), but they still promote DNA adsorption into the mesopores. This should be attributed to the observation that DNA can generate more hydrogen bonds within mesopores,27 making the adsorption process of DNA with M-MSNs occur under lower salt concentrations. Besides changing the salt concentrations, varying pH values also leads to the different adsorption behaviors for DNA with SNs and M-MSNs. As shown in Figure 2C and Figure 3C, when the pH values decreased from 6.8 to 4.1, the amount of adsorbed DNA increased moderately for M-MSNs, but changed negligibly for SNs. Because decreasing pH value is propitious to the generation of intermolecular hydrogen bonds,34 a following issue is whether the DNA in mesopores can form more hydrogen bonds and thus leads to a higher desorption energy barrier under lower pH values. According to the normalized curves shown in Figure 7B, at the same temperatures, the desorption ratios of the three M-MSN samples are very similar, suggesting analogous energy barriers in the desorption processes. Consequently, although decreasing pH values enhances DNA adsorption into M-MSN mesopores, the desorption barrier that is associated with the intermolecular hydrogen bonds changes negligibly.

(Supporting Information, Figure S2B). When the salt concentration increases, the Debye length decreases steadily. Once the Debye length becomes shorter than the length of hydrogen bonds, DNA will interact with silica surface through generating intermolecular hydrogen bonds (Supporting Information, Figure S2C). Unlike the process of DNA adsorption onto a SN surface, a prerequisite for DNA adsorption into M-MSN mesopores is the diffusion of DNA molecules into the inner space of mesopores. Figure 8B shows the cross section of mesopores with a cylinder-like structure. The negative charges on silica form an annular electrostatic field surrounding the internal surface, whose thickness is dominated by the Debye length in solution. When the Debye length becomes shorter than the pore radius, an electrostatic screening area (the region of the inside circle with curve slide) appears in the mesopore. As negative charge also exists on the DNA backbone, the electrostatic repulsion prohibits the sorbent diffusing into the mesopores, unless the screening region becomes large enough to accommodate the DNA molecule. Accordingly, the mesopores with smaller pore size require shorter Debye length, that is, higher salt concentration to generate such screening region for DNA entering the inner space of such mesopores. Because the pore diameter of M-MSNs has a distribution interval from ∼2 to ∼4 nm (shown in the Supporting Information, Figure S3), it seems that abundant mesopores with small pore diameter participate in the adsorption process when the salt concentration increases thus raising the adsorbed amount of DNA. On the basis of the above hypothesis, we further analyze the states of DNA within mesopores with different pore sizes. Fujiwara and co-workers26 have reported that DNA molecules existing in mesopores generated both direct and indirect hydrogen bonds (with four monolayers of water molecules between DNA and silica surfaces), which were associated with the pore diameters. Accordingly, a cross section of DNA in the mesopore with small pore size is described in Figure 8C. The screening area in such a mesopore has a similar diameter with the DNA molecule. Direct hydrogen bonds generate between DNA and the wall of the mesopore. In parallel to this, Figure 8D depicts the cross section of DNA in a mesopore with larger pore size. The diameter of the screening area in this mesopore is much larger than that of the DNA molecule (the location of DNA within mesopores was randomly drafted). Besides the formation of direct hydrogen bonds, DNA also generates indirect hydrogen bonds because the distance between DNA and the wall of the mesopores was too far to form direct hydrogen bonds. As the energy of hydrogen bonds has a negative correlation with the bond length, it seems that the energy of indirect hydrogen bonds for DNA adsorbed in large mesopores is lower than that of direct hydrogen bonds for DNA existing in small mesopores. In our previous work,27 we investigated the desorption behaviors of DNA from M-MSNs and SNs at different temperatures, and the result suggested that the energy barrier for DNA desorption was determined by the intermolecular hydrogen bonds between DNA and silica surface. Consequently, it is highly possible that the DNA desorption from large mesopores will be much easier than that from small mesopores at the same temperatures. This conclusion matches well with the result of our study about DNA desorption from three types of M-MSNs with varying average pore diameters. As shown in Figure 6B, at the same temperatures, the M-MSN sample with the average pore

5. CONCLUSIONS In this paper, the DNA adsorption capacities of M-MSNs were determined under aqueous solution conditions with changing the salt concentrations, the types of salt, and the pH values, respectively. Accordingly, further analysis suggested that the main driving forces for such adsorption process were the shielded electrostatic force, the dehydration effect, and the intermolecular hydrogen bonds. Although the types of driving forces were the same as those of DNA adsorption with solid silica nanoparticles, there were unique features for the interaction between DNA and M-MSNs aroused from the unique mesopore structures. When the salt concentration increased or the pH value decreased, the amount of adsorbed DNA grew up steadily, suggesting more mesopores participated into the DNA adsorption process. Further analysis depicted that the entrance of DNA into mesopores was probably controlled by the Debye length in solution. For DNA existing in mesopores with different pore sizes, the types of hydrogen bonds (direct or indirect) were determined by the mesopore diameter and thus resulted in different energy barriers for DNA desorption. In summary, this work provided deep insight into the mechanism of packaging DNA molecules within the MSN mesopores. Combined with our previous study, we have built a solid base for exploring MSN-based materials as delivery vehicles for nucleic acids.

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ASSOCIATED CONTENT

S Supporting Information *

Zeta potential of M-MSNs in various solutions with different salt concentration, sketches of the DNA adsorption onto SNs, and nitrogen sorption isotherms for three M-MSN samples with different average mesopore diameters. This material is available free of charge via the Internet at http://pubs.acs.org. 2833

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86-21-62933176; E-mail: [email protected].

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ACKNOWLEDGMENTS

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REFERENCES

This work was supported by National 863 High-Tech Program (2009AA03Z333), Shanghai Nano Project (1052 nm01100), and SJTU funding (YG2009ZD203, YG2010ZD102, AE4160003). The authors would like to thank the Instrumental Analysis Center of Shanghai Jiao Tong University for the characterization of materials. Especially, thanks to Miss Shufang He for the help in the isothermal titration calorimetric study and Mrs. Ying Dai for the graphic assistance.

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dx.doi.org/10.1021/la204443j | Langmuir 2012, 28, 2827−2834