Self-Assembled Functional Nanostructure of Plasmid DNA with Ionic

Apr 6, 2015 - (1) Generally in a few prokaryotic systems, such transformation can ... into DNA delivery vehicles, which are vectors that efficiently t...
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Self-Assembled Functional Nanostructure of Plasmid DNA with Ionic Liquid [Bmim][PF6]: Enhanced Efficiency in Bacterial Gene Transformation Sarvesh K. Soni,*,† Sampa Sarkar,† Nedaossadat Mirzadeh, P. R. Selvakannan, and Suresh K. Bhargava* Centre for Advanced Materials and Industrial Chemistry, School of Applied Sciences, RMIT University, GPO Box 2476, Melbourne, VIC 3001, Australia S Supporting Information *

ABSTRACT: The electrostatic interaction between the negatively charged phosphate groups of plasmid DNA and the cationic part of hydrophobic ionic liquid 1-butyl-3methylimidazolium hexafluorophosphate ([Bmim][PF6]), initiates spontaneous self-assembly to form the functional nanostructures made up of DNA and ionic liquid (IL). These functional nanostructures were demonstrated as promising synthetic nonviral vectors for the efficient bacterial pGFP gene transformation in cells. In particular, the functional nanostructures that were made up of 1 μL of IL ([Bmim][PF6]) and 1 μg of plasmid DNA can increase the transformation efficiency by 300−400% in microbial systems, without showing any toxicity for E. coli DH5α cells. 31P nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) spectroscopy, and X-ray photoelectron (XPS) spectroscopic analysis revealed that the electrostatic interaction between negatively charged phosphate oxygen and cationic Bmim+ tends to initiate the self-assembly process. Thermogravimetric analysis of the DNA-IL functional nanostructures showed that these nanostructures consist of ∼16 wt % ionic liquid, which is considered to provide the stability to the plasmid DNA that eventually enhanced the transformation efficiency.

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INTRODUCTION Bacterial gene transformation is one of the key techniques in molecular biology, wherein bacteria can receive a new genetic trait through a fragment of foreign DNA, and this method is having significant applications in gene cloning technology.1 Generally in a few prokaryotic systems, such transformation can happen naturally; however, most of the bacterial cells require artificial transformation, stemming from the slow diffusion of hydrophilic DNA’s entry across the hydrophobic lipid bilayer membrane and the slight electrostatic repulsion due to anionic DNA as well as the anionic headgroups of the bilayer membrane.2 Another possible disadvantage is that DNA can undergo hydrolysis or be degraded by any enzymatic process during its course of transformation.3 One way to circumvent this issue is to make the target cells more competent, which enables these cells to increase their uptake of genetic material.2 In parallel, the DNA molecules can be loaded into DNA delivery vehicles, which are vectors that efficiently transfer these genetic materials across the bilayer membrane and prevent any enzymatic damage to the DNA molecules. In this context, many DNA delivery vectors have been developed, and these methods mainly involve the encapsulation of DNA within the cationic surfactant, triblock copolymer vesicles, viral capsids, protein superstructures, lipid assemblies, polymer nanocapsules, and mesoporous structure, followed by transformation studies.4−9 In all of these cases, DNA undergoes a self-assembly process © 2015 American Chemical Society

with one of the delivery vectors and is crafted within the selfassembled superstructure. Even though all of these available techniques are simple and can be used for a broad range of species, there are still some limitations such as difficulties in a single-copy transgenic event, the need for expensive equipment and microcarriers, random intracellular targeting, and DNA damage due to its unmodified forms. Apart from this DNA delivery vehicle-mediated transformation, there are also some physical techniques such as a biolistic method, microinjection, electroporation, and heat shock, and poly(ethylene glycol)1,10−12 has also been used during the transformation to increase the efficiency. In particular, the well-established calcium chloride method followed by heat shock treatment has been used in studying the transformations using competent cells as targets. It is proposed that these divalent cations (Ca2+, Mg2+) first bind with anionic DNA and that these DNA molecules are transferred via the transient pores formed during the heat shock. In these cases, these ions form a salt with DNA, which enables efficient transformation. Despite the fact that these divalent cations interact with DNA due to their opposite charges and induced aggregation, they also require heat shock treatment in order to push the DNA molecules through the cell Received: February 3, 2015 Revised: April 1, 2015 Published: April 6, 2015 4722

DOI: 10.1021/acs.langmuir.5b00402 Langmuir 2015, 31, 4722−4732

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made up of DNA and IL were not affected by repeated washing, clearly indicating the strong interaction between DNA and IL molecules. The functional nanostructures of DNA-IL have been characterized by multiple techniques, including agarose gel electrophoresis as shown in Figure 1. On the agarose gel, DNA

wall. However, these inorganic cations bind to the DNA due to the complementary charges, but they do not direct any selfassembly. Rather, organic molecules containing cationic functional groups provide electrostatic interaction due to their strong opposite charge, and their interaction can induce the self- assembly process as a result of their hydrophobicity. In this context, using an ionic liquid (IL), consisting of an organic cation such as imidazolium and an inorganic anion, acts as a source of organic cations for the aforementioned process. Ionic liquids are stable at room temperature, less volatile, and less flammable and are used as green solvents for a wide range of organic and inorganic chemical transformations. By varying the substituent groups in the cation and anions in an ionic liquid, there are a number of physical, chemical, and electrochemical properties that can be varied and a number of biological applications can be started by exploring the use of ionic liquids as their designer solvents. One of the recent applications14 of using different kinds of ionic liquids in facilitating the polymerase chain reactions (PCR) of DNA escalated our interest in using ionic liquid-mediated bacterial transformation. Moreover, ionic liquids have also been used in the extraction of pure dsDNA without the addition of any extra material.13 On the basis of these studies, it is clear that IL can interact with DNA and that these hybrid materials can be potential nonviral synthetic vectors for enhancing the transformation process. This article reports the self-assembled nanostructures formed by the interaction of DNA and IL and their ability to enhance the transformation efficiency of plasmid DNA into the bacterial cell. We have shown functional nanostructures made of DNA and IL of various compositions and have demonstrated that for appropriate compositions these structures tend to increase the transformation efficiency of plasmid DNA in a bacterial cell. These nanostructures were demonstrated to be a stable, functional, and protected way to deliver DNA against any biological degradation. 1-Butyl-3-methylimidazolium hexafluorophosphate [Bmim][PF6] hydrophobic ionic liquid was chosen to form selfassembled nanostructures with plasmid DNA of various ratios. The bacterial pGFP gene transformation efficiency of these nanostructures was compared to that of pure DNA to determine the efficacy of these nonviral vectors in a bacterial transformation. Interestingly, these nanostructures retain the conformation (B form) of plasmid DNA and were found to enhance the transformation as compared to that of pure plasmid DNA, which clearly showed that these nanostructures can enhance the rate of uptake of the DNA molecule into the cell and protect the DNA from physicochemical mechanism. DNA-IL nanostructures of a certain composition were found to enhance the efficiency of transformation by 3- to 4-folds as compared to that of pure plasmid DNA. The DLS, XPS, 31P NMR, and FTIR studies clearly revealed that the interaction of the cationic 1-butyl-3-methylimidazolium (Bmim+) groups from the ionic liquid and the negatively charged phosphate group oxygen in plasmid DNA drive the self-assembly process.

Figure 1. Bands (under UV) of DNA and the DNA-Bmim+ (DNA+ IL) nanostructure in agarose gel (1%) electrophoresis showing no difference in plasmid DNA size and conformer.

fragments were separated according to their size; i.e., the rate of migration is proportional to size. Because DNA possesses negative charge, it tends to migrate toward the positive end. After the same amounts of plasmid DNA (40 ng) and DNAIL nanostructures have been loaded on gel, it has been observed that both bands moved to the same position. This suggests that although the DNA-Bmim+ nanostructure formed due to weak electrostatic interactions and Bmim+ did not neutralize the negative charge, DNA does not change its original functional property considerably. The separation of charge on the gel is same, and no retardation has occurred in the migration. Retaining the surface negative charge is important during the uptake of this genetic material. In general, Ca2+/Mg2+ ions mediated the DNA transformation; these metal ions neutralize the negative charge, which requires heat shock for the transformation. Cytotoxicity and Transformation Studies of DNA-IL Nanostructures on E. coli Cells. Prior to the transformation studies, the cytotoxicity of a pure ionic liquid on noncompetent bacterial cells was determined to estimate the threshold concentration of IL that did not show any significant toxicity to the cells. Figure 2A shows that the number of colonies formed was essentially the same in the absence of ionic liquid (a) and in 5 μL of IL (b). These results indicate that for up to a 5 μL quantity of ionic liquid, no toxicity was found. Subsequently, the role of varying amounts of ionic liquid with a constant amount of DNA and their effect on the transformation efficiency were studied. The DNA-IL nanostructures were prepared by varying the volume of IL from 0 to 10 μL while maintaining the total amount of DNA (10 μL of solution from a 100 μg/mL DNA solution) and the total volume constant. Figure 2B(a−d) shows optical images of the transformed cells, when the volume of the ionic liquid was kept at 0, 0.5, 1, and 5 μL for the same concentration of DNA. The

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RESULTS AND DISCUSSION Synthesis of DNA-IL Functional Nanostructures and Agarose Gel Electrophoresis. Aqueous solution of plasmid DNA was mixed with ionic liquid [Bmim][PF6] and incubated on a high-speed vortex mixer overnight. The resultant material was washed vigorously with ethanol and acetonitrile (1:1) in order to remove free ionic liquid; however, the nanostructures 4723

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Figure 2. (A) Number of colonies of noncompetent E. coli DH5α cells (200 μL of 1 × 106 cells/mL) formed in the (a) absence and (b) presence of 5 μL of ionic liquid [Bmim][PF6]. (B) Optical image of transformed E. coli cells (green fluorescent protein expressed, under UV illumination), showing the effect of increasing volume of ionic liquid: (a) 0, (b) 0.5, (c) 1, and (d) 5 μL. (C) Number of transformed colonies as a function of the volume of ionic liquid (using 10 μL of DNA from a 100 μg/mL concentration).

number of transformed colonies formed in the case of 1 μL of IL was much higher, while the number of transformed colonies in the case of 5 μL of IL was found to be very low. The stable transformed fluorescent colonies seen on the agar plate with added antibiotic clearly showed that 0.5−1 μL of ionic liquid showed the maximum (300−400%) transformation efficiency. Figure 2C shows the number of transformed colonies formed as a function of the volume of IL (in μL) used for the transformation. It is clearly seen that a rapid enhancement in transformation efficiency was observed when a much smaller volume of ionic liquid was used, and it started to decline at a higher IL volume. These results showed that the ionic liquid played a vital role in modulating the transformation efficiency. This volume is much less than the 5 μL threshold concentration of IL above which it becomes toxic to noncompetent bacterial cells. It is well known that the cation part of IL tends to interact with the negatively charged DNA.13 Therefore, the addition of a hydrophobic IL such as [Bmim][PF6] to a plasmid DNA tends to hydrophobize the DNA, and the effect may be stronger at higher concentrations of IL. Increased hydrophobicity of the plasmid DNA as a result of its interaction with greater amounts of IL (more than 1 μL) may inhibit its diffusion in a polar medium and its uptake in the hydrophilic cell membrane of E.coli. From the previous results, it is understood that 1 μL of ionic liquid underwent a very high transformation; therefore, DNAIL nanostructures of increasing concentration of DNA were

prepared while keeping the volume of the ionic liquid constant (1 μL). As a control, the same amount of plasmid DNA was used for the transformation, and the transformation efficiency of pure plasmid DNA and DNA-IL nanostructures is given in Figure 3. The agar plate with added antibiotic shown in Figure 3A clearly represents the number of colonies formed in the absence and in the presence of IL, when the DNA concentration gradually increased. Clearly, the DNA-IL nanostructures had a very high transformation efficiency with respect to plasmid DNA (Figure 3). The amount of DNA (μL) against the number of colonies formed for pure plasmid DNA and DNA-IL nanostructures is plotted as Figure 3B. In both cases, the transformation increased in a sigmoidal pattern of growth; however, pure plasmid DNA has a lower transformation efficiency as compared to that of DNA-IL nanostructures at all DNA concentrations. Figure S1 presents the plates with transformed colonies of E. coli. with 2−200 μL of plasmid DNA (both interacting and noninteracting with IL). Figure 4 shows a graphical illustration of the transformation efficiency as a function of DNA concentration in the presence and absence of IL. The maximum difference in transformation efficiency occurred for up to 20 μL (8 ng) (0.4 μg/mL) of DNA-IL nanostructures, after which the enhancement became saturated. These results indicate that it is possible to enhance the transformation efficiency to up to 300−400% of the functional DNA-IL nanostructures as compared to pure plasmid DNA, 4724

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between plasmid DNA and [Bmim][PF6], 31P NMR analysis of pure plasmid DNA, IL, and DNA-IL nanostructures was carried out and is shown in Figure 5A. The hexafluorophosphate anion present in the IL showed its characteristic heptet (due to P−F coupling) peak in the upfield region ranging from −125.2 to −163.2 ppm (Figure 5A, spectrum a), and phosphate and the phosphodiester groups present in the plasmid DNA showed their chemical shift at −0.68 ppm (Figure 5A, spectrum b). The DNA-IL functional nanostructures (Figure 5A, spectrum c) exhibit two phosphorus chemical shifts corresponding to both the DNA phosphate (P−O−P) and PF6 ions present in the IL; however, only DNA phosphate groups showed an upfield shift from −0.68 to −1.11 ppm whereas there was no change observed in the heptet peak that corresponds to the [PF6]− ions. This clearly suggests that the negatively charged phosphate groups are the site of interaction with the cationic part of the IL. Similar results were observed in the 31P NMR spectra of the sodium salt of DNA interacting with an ionic liquid.13 NMR analysis of these materials showed that the interaction between the Bmim cation and negatively charged phosphate groups from the DNA may be a direct consequence of the electrostatic-interaction-mediated self-assembly process. Due to the intense [PF6] chemical shift, the number of IL units present in the nanostructures must be greater than the number of DNA units, and it indirectly proves that many Bmim cations interact with more than one phosphodiester group of one DNA molecule. More clear evidence regarding the interaction of DNA and IL comes from the FTIR spectral analysis of these materials because IL and DNA have distinct vibrational features and any changes observed in the FTIR spectrum can be correlated to the functional groups’ interaction. FTIR spectra of IL, plasmid DNA, and DNA-IL nanostructures are shown in Figure 5B (curves a−c, respectively), and the results are in agreement with the NMR results that were shown in the previous section. On the basis of the electrophoresis and NMR studies, we have concluded that [Bmim]+ preferentially interacts with DNA, but FTIR analysis of these materials provided further understanding of the functional groups’ interaction. In FTIR spectra, the symmetric stretching of P−O bond vibrations in the phosphate group (P−O) bond observed at 1045 cm−1 was assigned to the B form of DNA.15 Thus, the unchanged symmetric stretching vibrational band at 1045 cm−1 suggested that this kind of interaction happened only on the surface of a DNA molecule, which does not alter the helical structure of DNA (B form). This shows that the B-form conformation of DNA was retained within the DNA-IL nanostructures. Studies have already shown that cationic species such as metal ions can bind to the phosphate groups of DNA strongly and change their conformation, which eventually results in changing the position and intensity of the bands.16−18 Furthermore, one of the recent studies reported that the conformation of the B form of DNA did not change when it coexisted with the Bmim cation in an ionic liquid.13 In the case of DNA-IL nanostructures, the symmetric stretching vibrational band of P−O at 1045 cm−1 is almost unaffected (Figure 5B curve b and Figure 5B curve c); however, the band at 1084 cm−1 in Figure 5B curve c became very prominent (observed as a small hump in curve b). The asymmetric P−O vibration observed at 1084 cm−1 may be due to its interaction with Bmim+.19 Figure 5B(a) corresponds to the FTIR spectrum of pure ionic liquid [Bmim][PF6], which

Figure 3. (A) Optical image of transformed E. coli cells (green fluorescent protein expressed, under UV illumination) using increasing volumes (2−30 μL) of plasmid DNA (100 μg/mL) in the absence and presence of IL [Bmim][PF6]. (B) Graphical presentation of the effect of different volumes (2−200 μL) of DNA (400 ng/mL) (with and without IL) on the transformation of 200 μL of 1 × 106 E. coli. cells/ mL.

when 8 ng of DNA and 1 μL of IL was used to make functional nanostructures. These transformation studies have clearly shown that the nanostructures made up of DNA-IL can substantially improve the transformation efficiency, so it is important to study the nature of interaction between DNA and IL to elucidate the enhancement in efficiency. 31 P NMR and FTIR Studies of DNA-IL Functional Nanostructures. To understand the nature of the interaction 4725

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Figure 4. Comparison of the transformation efficiency of the DNA-IL nanostructure and pure DNA on competent E. coli cells with increasing amounts of DNA (0.8−80 ng).

estimated to be 55.2 nm, and after interaction with an ionic liquid the hydrodynamic radius of the DNA-IL complex increased to 69.5 nm (Figure 6A).20 The change in the radius of DNA is due to the strong electrostatic binding of the Bmim cation with the DNA, and the incorporation of IL molecules on the surface of DNA might be responsible for the increase in the hydrodynamic radius. Such electrostatic interactions and inclusion of IL molecules can favor the formation of DNA-IL nanostructures without exerting any significant structural changes on DNA. In an earlier study, cryo-TEM imaging proved that the cationic Bmim+ groups not only bound parallel to the surface but also arranged on the surface of the DNA, due to which the conformation of the B form of DNA has been changed.20 Therefore, the current results agree very well with the earlier observation, and TEM imaging of plasmid DNA after its interaction with IL was performed to study the morphological changes and the images are presented as Figure 6. The TEM images of Figure 6B(a,b) show the average diameter of plasmid DNA in the solid phase to be about 20−30 nm, and after interaction with IL, the average diameter of the DNA-IL nanostructures increased up to 50−60 nm, respectively. These results support the fact that the incorporation of IL molecules on the surface of DNA is responsible for the increase in the size of the structures observed in the TEM images. TEM imaging provided information that supercoiled plasmid DNA tends to forms a coiled structure during the sample preparation, so the average diameter was found to be lower than the average diameter estimated from DLS. CD Spectroscopy and TGA Analysis of DNA-IL Functional Nanostructures. It is important that the secondary structure of plasmid DNA undergoes changes after its interaction with IL because certain conformations of DNA are not acceptable during the transformation, and the B form of plasmid DNA is a suitable conformer for transformation studies. CD spectroscopy is primarily used in studies of secondary structure of DNA,20−22 hence CD spectra of DNA and the DNA-IL nanostructure was obtained and are shown in Figure 7A. The CD spectrum of pure plasmid DNA shows a broad, positive wave band at 269 nm, but the spectrum of DNA-IL nanostructures shows an intense, long wave positive band at the same wavelength. The broad CD spectrum of plasmid DNA was attributed to the contributions of various degrees of freedom; however, all conformations are the B form of DNA. Interestingly, DNA-IL nanostructures showed an

Figure 5. (A) 31P NMR spectra (a) [Bmim][PF6], (b) pure plasmid DNA, and (c) DNA-IL nanostructures. (B) FTIR spectra of (a) [Bmim][PF6], (b) pure plasmid DNA, and (c) DNA-IL nanostructures.

showed an intense peak at 1180 cm−1 that was assigned to the Bmim ring asymmetric stretching vibration. This peak was absent in the case of DNA-IL nanostructures, as shown in Figure 5B(c). Therefore, FTIR and NMR spectral analysis revealed that the DNA-IL nanostructure formed as a consequence of the electrostatic interaction between Bmim groups and IL molecules and this interaction might be responsible for the formation of the nanostructures.20 DLS and TEM Analysis of the DNA-IL Functional Nanostructures. Dynamic light scattering was used to study the changes in the hydrodynamic radius of plasmid DNA before and after its interaction with the ionic liquid. DLS studies revealed that the hydrodynamic radius of plasmid DNA was 4726

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Figure 6. (A) Dynamic light scattering studies of DNA and DNA-IL nanostructures. (B) TEM images of plasmid DNA (a) and DNA-IL nanostructures (b).

Figure 7. (A) CD spectra of pure DNA and DNA_IL nanostructures. (B) Thermogravimetric analysis curves of [Bmim][PF6], pure plasmid DNA, and DNA-IL nanostructures.

intense CD spectrum at the same wavelength, indicating that IL and Bmim cations bind strongly to the anionic phosphate groups, and this strong interaction led to a restriction of DNA to its native conformation (B form).20 We studied the thermal stability of the DNA-IL nanostructures in order to evaluate the strength of the binding between DNA and IL, thus thermal gravimetric analysis (TGA) of pure plasmid DNA and the DNA_IL nanostructure was carried out

and is shown in Figure 7B. It is well known that pure IL is basically a molten salt and has a very high boiling point due to the strong electrostatic interaction between cation Bmim and anion PF6. Hence, IL showed thermal stability up to 400 °C but was completely decomposed to within the temperature range of 400−500 °C. Pure DNA shows continuous weight loss due to the evaporation of water, decomposition of sugars, and bases, and the overall weight loss was around 55% up to 600 °C, 4727

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Figure 8. XPS core-level spectra recorded from the pure DNA and DNA after interaction with ionic liquid [Bmim][PF6]. (a) O 1s core-level spectra. (b) P 2p core-level spectra. (c) N 1s core-level spectra. (d) C 1s core-level spectra from DNA and DNA-IL nanostructures. The solid lines correspond to nonlinear least-squares fits to the experimental data as shown by symbols.

XPS Analysis of the DNA-IL Nanostructures. XPS analysis of pure DNA and DNA-IL nanostructures was further carried out to investigate the nature of interactions.24 Figure 8 shows the N 1s, P 2p, O 1s, and C 1s core-level spectra of pure plasmid DNA and the DNA-Bmim+ nanostructure. The P 2p core-level spectra of pure plasmid DNA and DNA-IL nanostructures are given in Figure 8b. In the case of plasmid DNA, its shows only one spin−orbit pair at a binding energy (BE) 133.6 eV that corresponds to the phosphorus present in the phosphate groups of DNA. However, the as-formed DNA-

leaving some residual carbon and phosphate groups. As compared to pure DNA, the DNA-IL complex started decomposing in a higher temperature range, clearly suggesting that the stability of DNA was increased after the incorporation of IL. However, in the temperature range of 250−450 °C, the DNA-IL complex shows a sharp weight loss, probably due to the loss of Bmim cations bound to DNA. In addition to that, the overall weight loss of the DNA-IL complex at 600 °C was 16% higher than that of natural DNA, suggesting that at least 16% of the IL must be bound to DNA.23 4728

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Scheme 1. Hierarchical Assembly of Purified pGFP Plasmid DNA from Recombinant E. coli DH5α with Ionic Liquid [Bmim][PF6] and the Electrostatic Interaction of the [Bmim]+ Ion and PO4− of the DNA Strand, Leading to the Synthesis of a Functional, Stable DNA-IL Construct

532.7 eV, which were assigned to the oxygen atoms present in the phosphate and deoxyribose sugar groups, respectively. The relative intensity of the O 1s coming from the sugars [BE 532.7] was found to be slightly more intense than that of the O 1s component from the phosphate oxygen’s [BE 531.3] two components. In the case of DNA-IL nanostructures, both components have shown a 0.2 eV shift toward higher binding energies, and interestingly, their relative intensities changed significantly. The relative intensity of phosphate oxygen groups was found to be more intense [BE 531.5] than the intensity of the O 1s component coming from the sugar units [533 eV], which is exactly opposite to the case of pure plasmid DNA O 1s component spectra. Because XPS is a surface-sensitive technique, the results indicate that there will be higher percentage of phosphate units than sugar units in the surface of DNA-Il nanostructures. This may happen when DNA interacts with the IL molecules through its phosphate groups, and these groups might be more-commonly located on the surface of a spherical DNA-IL nanostructure with their sugar units facing inward. Thermal gravimetric analysis has also proven that 16% of the ionic liquid was incorporated into this nanostructure. In particular, the intensity of the oxygen 1s levels corresponds to the increased level of phosphate oxygen, and the peak width increases considerably. This shows that phosphate oxygens could be the preferred binding sites for the ionic liquid. In Figure 8c, the N 1s core-level spectra of DNA and the DNABmim+ complex reveal more information regarding the interaction between DNA and [Bmim][PF6]. Plasmid DNA has three distinct N 1s components at 398.8, 399.9, and 401.6 eVs, which were attributed to the DNA bases. After the formation of DNA-IL nanostructures, the relative intensity of the high BE N 1s component at 401.8 eV increased significantly. This will happen only when the Bmim+ cation is incorporated inside the structures because the cationic heterocyclic nitrogen of Bmim+ usually shows its N 1s components in this region. Becasue we have not seen many changes in the BEs of DNA bases, this led to the conclusion that hydrophobic DNA bases were not participating in this

IL nanostructures exhibit two different spin−orbit pairs at 133.6 and 136.7 eV, which were assigned to the phosphorus atoms present in the phosphate groups of DNA and the hexafluorphosphate groups from [Bmim][PF6]. This is in agreement with the NMR results that also showed the presence of [PF6] groups. The presence of [PF6] groups even after extensive washing of the obtained assembly (DNA-Bmim+) within the aqueous phase clearly shows that Bmim+ cations were incorporated with the DNA and the presence of [PF6] ions was to provide electroneutrality. This also indicates that the Bmim+ cation did not form any strong interaction with the DNA or an ion pair with DNA. Instead, it form a weak electrostatic interaction with the phosphate groups of DNA, and that may be the reason that the DNA retained its conformation. In the case of DNA-IL nanostructures, the P 2p core-level component that corresponds to the PF6 ions [BE 136.7 eV] was much more intense than that of the P 2p component that corresponds to the DNA phosphorus [BE 133.6]. The relatively high intensity of the PF6 groups versus the DNA phosphate groups indicates that there will be a significant number of Bmim+ cations present in the complex on the surface of the DNA. Therefore, the DNA-IL nanostructures may be formed as a result of multiple weak electrostatic interactions between the IL molecules and phosphate groups present over the entire plasmid DNA. Another interesting observation is that there was not much change in the BE [133.6 eV] of DNA phosphate groups, leading to the conclusion that the phosphorus ends of phosphate ions were not the binding sites for Bmim+ cations. However, significant changes in the O 1s core-level spectra observed in the case of pure plasmid DNA and after its interaction with [Bmim][PF6] and the O 1s core-level spectra are shown in Figure 8a. Because IL [Bmim][PF6] does not contain any oxygen, any changes observed in the case of the O 1s core-level spectra were a direct consequence of any modification occurred in the DNA phosphate or sugar units. In the case of pure plasmid DNA, O 1s core-level spectra were resolved into two main components centered at 531.3 and 4729

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Isolation of Plasmid DNA. A pGFP plasmid preparation from E. coli DH5α was obtained on the basis of the alkaline-SDS method.25 A transformed bacterial culture (50 mL) was centrifuged, and the cell pellet was suspended in 3 mL of TE-glucose [50 mM glucose, 10 mM EDTA, 25 mM Tris−HCl (pH 8.0)]. The suspension was vortex mixed properly. To this, 8 mL of an alkaline-SDS solution (0.2 N NaOH, 1% SDS) was added, and the mixture was mixed gently and incubated for 10 min, following which it was neutralized by adding 8 mL of 3 M potassium acetate (pH 4.8) and was then centrifuged at 12 000 rpm for 10 min at 4 °C. The supernatant was treated with chloroform−isoamylalcohol (24:1). The upper layer was collected, and to this 2 volumes of absolute ethanol was added, following which the mixture was subjected to further centrifugation at 14 000 rpm for 10 min. The pellet was rinsed with 70% ethanol, again centrifuges at the same speed, dried, and dissolved in 50 μL of TE buffer. E. coli DH5α Culture and Competent Bacterial Cells. E. coli DH5α colonies were picked and transferred to 10 mL of LB in 50 mL conical glass flasks (starter cultures) and were incubated aerobically at 37 °C/200 rpm for 24 h. The inoculums of 1 mL were transferred into four conical glass flasks (250 mL) containing 50 mL of LB and incubated aerobically at 37 °C/200 rpm for 3 h (OD600 0.6−0.8) to obtain exponential phase cells. At the start of the experiment, the inoculated broth was kept in an ice bath for 30 min. Cultures were pelleted down by centrifugation at 4 °C/4500 rpm for 10 min. We resuspended the whole pellet in 2 mL of LB and made the cells competent by the following standard calcium/magnesium chloride method, which is the procedure described by Zhiming et al.26 Exponentially growing cells ( 200 μL, 1 × 106 cells/mL) were pelleted down by centrifugation at 4 °C/4500 rpm for 10 min. The pellet was resuspended in an ice-cold filtered solution of 1 mL of MgCl2 (80 mM)/CaCl2 (20 mM), mixed gently, and again pelleted down by centrifugation at 4 °C/4500 rpm for 10 min following the same steps. The final pellet was resuspend in 200 μL of ice-cold CaCl2 (100 mM) solution and kept in an ice bath. Streptomycin was added to an LB agar plate (10 μg/mL) for the transformation experiments. E. coli DH5α PGFP transformants were grown in LB medium supplemented with 10 μg/mL streptomycin. Transformation of Competent Cells. Competent bacterial cells were transformed with DNA by following the method described by Douglas’ group.27 Freshly prepared competent cells incubated with plasmid DNA and the plasmid DNA-Bmim+ complex gel at various volumes (0.5−200 μL) from a 1:10 dilution of 1 mg/mL stock solution for 30 min in an ice bath and was then heat shocked at 42 °C for 90 s and kept in an ice bath for 2 min. LB (800 μL) was added and kept on an orbital shaker at 37 °C/200 rpm for 1 h. This culture (100 μL, 1:10 dilution) was plated and allowed to sit overnight. E. coli DH5α PGFP transformants were grown in an LB agar plate medium supplemented with 10 μg/mL streptomycin. Transformation Efficiency. To determine the transformation efficiency of the stable transformation, competent cells were transformed as described above, and we calculated the colony-forming unit (CFU) per nanogram of only plasmid DNA and the plasmid DNA-[Bmim+] complex. In the presence of the DNA-[Bmim+] complex, an enhancement of the transformation efficiency was shown on the LB agar plate medium supplemented with 10 μg/mL streptomycin by capturing images with an optical digital camera. Amalgamation and Characterization of the Plasmid DNAIonic Liquid ([Bmim][PF6]) Complex. Plasmid DNA (10 μL, 16 mg/mL) was mixed with 990 μL of [Bmim][PF6] in a 1.5 mL microcentrifuge tube and held for overnight vortex mixing. The reaction mixture was then centrifuged at 14 000 rpm for 30 min, and the supernatant was removed and pallet was washed twice with an ethanol (50%, semichilled) and acetonitrile (50%) mixture. The gellike plasmid DNA-[Bmim]+ complex was observed by washing with acetonitrile (100%). We dissolved the gel in distilled water, and made the final concentration 1 mg/mL; 5% of the DNA was lost during removal of excess [Bmim][PF6] ionic liquid. [Bmim][PF6] interacted with the DNA surface; i.e., [Bmim][PF6] was bound to the DNA entity.

electrostatic interaction. On the other hand, no significant change has occurred in the C 1s core-level spectra, which has been shown in Figure 8d. Thus, this clearly indicates that due to the strong electrostatic interaction between DNA and [Bmin][PF6], the DNA-Bmim+ nanostructure has been formed, which does not affect the B structure of DNA. All of the aforementioned physiochemical characterization of the DNA-IL nanostructures clearly depicts that the electrostatic interaction between the negatively charged phosphate oxygen and the cationic Bmim led to the self-assembly process to form the functional DNA-IL nanostructures. These structures retained the natural conformation of DNA, and when these structures were used as nonviral synthetic vectors, they showed a very high enhancement for bacterial cell transformation. The enhancement was due to the increased stability of DNA within the DNA-IL nanostructures, their protection of DNA against any enzymatic degradation, and the higher rate of diffusion due to increased charge density around the DNA surface. On the basis of these studies, a simple model is proposed and given as Scheme 1 to provide the fundamental interaction between DNA and hydrophobic IL and their role in enhancing the bacterial transformation efficiency.

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CONCLUSIONS A simple approach of synthesizing DNA-IL functional nanostructures and their use as synthetic nonviral vectors for the efficient bacterial pGFP gene transformation in cells has been demonstrated. Electrostatic interaction between hydrophobic ionic liquid [Bmim][PF6] and plasmid DNA led to the formation of self-assembled nanostructures, wherein the Bmim cation was found to interact with the negatively charged phosphate oxygen groups. DNA-IL nanostructures with varying compositions of IL and DNA were prepared and screened for their transformation efficiency. In particular, the nanostructures that consist of small concentrations of IL tend to enhance the transformation efficiency by up to 300−400%. 31P NMR, FTIR, and XPS analysis revealed the nature of the interaction between the Bmim cations and the phosphate oxygen groups. Circular dichrosim analysis of the DNA-IL nanostructures indicated that the B conformer of DNA was retained within the nanostructures, and this conformation is essential for efficient transformation. TGA analysis has shown that these nanostructures consist of up to 16 wt % of the IL molecules, which provide stability to the plasmid DNA nanostructures. The selfassembly of IL molecules on the surface of plasmid DNA protects the DNA against any enzymatic degradation and provides enough charge density to transfer across the cell membrane. Overall, the self-assembled nanostructures of plasmid and a hydrophobic ionic liquid were demonstrated to be promising nonviral vectors for gene transformation, and this is clearly one of the earlier studies in the application of DNA-IL functional nanostructures in bacterial transformation. We will further explore the area of molecular biology utilizing task specific ionic liquids.

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EXPERIMENTAL SECTION

Chemicals. Ionic liquid (IL) 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF6]) was purchased from Ionic Liquid Technologies (IoLiTec). Luria-Bertani (LB) broth, CaCl2, and MgCl2 were purchased from Sigma-Aldrich. E. coli DH5α and a recombinant strain with pGFP plasmid were obtained from the School of Applied Sciences, RMIT University. 4730

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Langmuir Material Characterization. The 31P NMR spectra of DNA, [Bmim][PF6], and the DNA-Bmim+ adduct in water were obtained at 298 K in dimethyl sulfoxide (DMSO). NMR spectra were recorded on a Bruker Avance 300 spectrometer. The chemical shifts were measured relative to 99% orthophosphoric acid. The FTIR (Fourier transform infrared) spectra for plasmid DNA, [Bmim][PF6], and the DNABmim+ adduct in the water phase were recorded at the School of Applied Sciences Vibrational Spectroscopy Facility. IL was used as received, and the precipitated DNA/DNA-IL nanoconstruct was dissolved in water (0.1 mg/mL) and used as a droplet (10 μL) for the PerkinElmer Spectrum 100 with a universal single-bounce diamond attenuated total reflectance (ATR) attachment. The infrared spectra were obtained on a PerkinElmer Spectrum 2000 FTIR spectrometer. The DLS experiments were performed on an ALV two color crosscorrelation multiple scattering suppression spectrometer. The samples were prepared in filtered (with a 0.45 μm Millipore syringe filter) deionized water and diluted accordingly to get the optimal scattering data using quartz tubes. The CD experiments were performed on a Jasco J-815 circular dichroism (CD) spectropolarimeter in the range of 320−220 nm at a scanning speed of 50 nm/min using deionized water as the solvent (4% w/v). TEM imaging of DNA and the DNA-Bmim+ adduct was carried out by using a 100 kV JEOL 1010 TEM instrument. The DNA and DNA-IL nanoconstruct were precipitated out with chilled ethanol, dried, and further suspended in deionized water to be drop cast on a TEM copper grid (10 μL of a solution containing sample). The samples were air dried and imaged under an electron beam. The nature of the chemical interaction of DNA with [Bmim][PF6] characterized by XPS measurements was carried out on a Thermo KAlpha XPS instrument at a pressure of better than 1 × 10−9 Torr (1 Torr = 1.333 × 102 Pa). The general scan and N 1s, P 2p, O 1s, and C 1s core-level spectra of pure plasmid DNA and the DNA-Bmim+ adduct and core-level spectra from the respective samples were recorded with monochromated aluminum Kα radiation (photon energy = 1486.6 eV) at a pass energy of 20 eV and an electron takeoff angle (angle between the electron emission direction and surface plane) of 90°. The core-level binding energies (BEs) were aligned with the adventitious carbon binding energy of 285 eV.

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instruments used in this study. We thank Mr. Reece NixonLuke for his help in the DLS experiments. We also thank Dr. Shiv Shankar and Prof. Vipul Bansal for their contributions in preliminary discussions and valuable suggestions regarding the transformation experiments. We are also very grateful to Dr. Steven Priver for proofreading and correcting grammatical errors in this article.

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ABBREVIATIONS [BMIM][PF6], 1-butyl-3-methylimidazolium hexafluorophosphate; XPS, X-ray photoelectron spectroscopy

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ASSOCIATED CONTENT

S Supporting Information *

Transformation efficiency of DNA-IL nanostructures and images of GFP expressing transformed colonies with variable volumes of DNA-IL. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: +61 3 9925 2397. *E-mail: [email protected]. Tel: +61 3 9925 2330 Author Contributions †

S.K.S. and S.S. contributed equally to this work.

Author Contributions

The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS S.K.S. and S.S. thank the Australian Government, Department of Education, for an Endeavour Research Award and the School of Applied Sciences, RMIT University, for an ECR startup grant. We duly acknowledge the RMIT Microscopy and Microanalysis Facility (RMMF) for providing access to their 4731

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