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Comparison of nanocarriers for gene delivery and nanosensing using montmorillonite, silver nanoparticles and multiwalled carbon nanotubes
Applied Clay Science 103 (2015) 55–61
Contents lists available at ScienceDirect
Applied Clay Science journal homepage: www.elsevier.com/locate/clay
Note
Comparison of nanocarriers for gene delivery and nanosensing using montmorillonite, silver nanoparticles and multiwalled carbon nanotubes T. Anitha Sironmani ⁎ School of Biotechnology, Madurai Kamaraj University, Madurai 625021, India
a r t i c l e
i n f o
Article history: Received 8 March 2014 Received in revised form 4 November 2014 Accepted 5 November 2014 Available online xxxx Keywords: DNA functionalizing Silver nanoparticles montmorillonite MWCNT SPR based DNA sensor Nonviral gene vector
a b s t r a c t Various types of nanoparticles like montmorillonite, silver nanoparticles stabilized with montmorillonite, starch, citrate, polylysine and multiwalled carbon nanotubes were used for binding with plasmid pcDNA-GFP in order to develop a gene delivery vector. The characterization of the functionalized materials showed a shift in the plasmon resonance to a higher wavelength compared to the control after DNA binding. This revealed the possibility that the DNA can be detected as a change in the absorption probability and/or a change in the resonance wavelength which can be used for diagnostic purposes. Transfection studies of these various functionalized nanopreparations implied that the gene delivery vector based on silver nanoparticles stabilized with starch and montmorillonite were more promising than other gene delivery systems with the potential to revolutionize the area of biosensing, imaging, diagnosis and therapy. © 2014 Elsevier B.V. All rights reserved.
1. Introduction For therapeutics, vaccination, replacing defective genes, gene delivery, abnormal cell destruction and for tissue regeneration, gene delivery vectors with minimal toxicity offer great potentials (O'Connor and Crystal, 2006; Wang and Yuan, 2006). The immune response elicited by viral proteins poses a major problem, leading to the development of nonviral DNA delivery vehicles. Nanoscience is an extremely powerful emerging science, which is expected to have a substantial impact on medical science now and in the future. The overall efficacy of nanoparticles to act as potential non viral gene delivery vehicles depends on the physical properties of nanoparticles, including their morphology, size, charge density and stability (Glover et al., 2005; Jackson et al., 2006; Khalil et al., 2006). Recently, synthesis of silver nanoparticles has attracted considerable attention owing to their diverse properties like catalysis (Shiraishi and Toshima, 2000), magnetic and optical polarizability (Shiraishi and Toshima, 2000), biosensor (Nimrodh Ananth et al., 2011), electrical conductivity (Chang and Yen, 1995), antimicrobial activity (Sharverdi et al., 2007) and Surface Enhanced Raman Scattering (SERS) (Matejka et al., 1992). In recent years, emerging applications of carbon nanotubes have shown their promising potentials in biomedical fields including novel delivery systems for drugs or DNA/RNA (Beg et al., 2011). Nanosilver can be modified for better efficiency to facilitate its diverse applications in medicine and life sciences. In order to enhance the biocompatibility and cell affinity for gene delivery, various methods of nanosilver preparation to functionalize with plasmid DNA for gene ⁎ Tel.: +91 0452 2458905. E-mail addresses: [email protected], [email protected].
http://dx.doi.org/10.1016/j.clay.2014.11.004 0169-1317/© 2014 Elsevier B.V. All rights reserved.
delivery were attempted. The green materials used in this study included montmorillonite (Mt), silver nanoparticles stabilized in montmorillonite (Ag + Mt), silver nanoparticles synthesized and stabilized with starch (Ag + starch), citrate (Ag + citrate) and polylysine (Ag + polylysine) and for comparison Mt and multiwalled carbon nanotube (MWCNT) were also used. The plasmid DNA (pcDNA) vector conjugated with complementary DNA of green fluorescent protein as reporter gene was used as the model DNA/gene to compare the performance of nanoparticles as gene delivery vectors. Understanding the unique characteristics of engineered nanomaterials and their interactions with DNA and biological systems is key to the safe implementation of these materials in novel biomedical diagnostics and therapeutics. Clay minerals have interesting chemical and physical characteristics, e.g., Mt has a high modulus, high cation exchange capacity, a large surface area to mass ratio and the ability to form stable dispersions in aqueous solutions (Marzilli, 1977; Bergaya et al., 2006). Clay mineral has been used successfully as vectors for delivery of DNA into cells in recent experiments (Zhu et al., 2002; Kiruba Daniel et al., 2010). The current study paves the base line on the biocompatible methods of synthesis of silver nanoparticles that enhances their usage in medicine for diagnostics and therapeutics. 2. Methods 2.1. Synthesis of nanoparticles The silver nanoparticles were synthetized in the interlamellar space of a layered Mt (Kiruba Daniel et al., 2010). Ag + starch was prepared as
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described in earlier studies (Kiruba Daniel et al., 2011). Ag + citrate and Ag + polylysine were prepared by following the earlier methods (Rivas et al., 2001; Zhu et al., 2002). Activated MWCNT prepared by the method developed by Sun et al. (2002) were provided as a gift by Prof. Saraswathy, Material Sciences, School of Chemistry, M.K. University, Madurai, Tamil Nadu, India. 2.2. Preparation of nanoparticle–DNA complexes A commercial solution of plasmid DNA, a complementary DNA for green fluorescent protein (GFP) containing vector (pcDNA-GFP vector, Clontech, Palo Alto, CA) was functionalized to various nanopreparations at a concentration of 50 μg ml−1 at 37 °C for 2 h. The nanoparticles and DNA were mixed for 2 h with and without salt. The functionalized nanoparticles were centrifuged at 10,000 rpm for a minute and the DNA content of the supernatants was measured at A260. The amount of adsorbed DNA was calculated by subtracting the amount present in the supernatant from the amount initially added. 2.3. Characterization of nanoparticles UV–visible spectra were recorded on a Shimadzu UV 1700. All the fluorescence data were obtained on a Perkin-Elmer LS50B luminescence spectrometer, The excitation wavelength used was 380 nm. CD spectra of control and DNA functionalized nanoparticles were taken in aqueous solutions at pH 7 under normal temperature using a JASCO J-810 Spectropolarimeter. FTIR spectra of all the functionalized nanoformulations were recorded on a Bruker Fourier transform infrared spectrometer (Vector 22 model) in the range 400–4000 cm−1 with a resolution of 4 cm−1. 2.4. Transfection efficiency Liver cells (from Swiss mouse), secondary culture (after passage 10, established in the lab) were seeded in Petri plates at a density of 5000 cells per plate in antibiotic-free DMEM with 10% serum. The amount of nanoparticles with DNA equivalent to 0.5 μg was added to each plate after 24 h. The cells were then incubated at 37 °C, 5% CO2 for 4 days, after which the cells were observed under fluorescence microscope (Nikon Eclipse TE2000-U) at a wavelength of 490 nm. Transfection with calcium phosphate-DNA was done as positive control. All transfection experiments were performed in triplicate. The transfection efficiency was calculated using the fluorescence expressed from the plasmid DNA encoding the GFP gene in pooled samples. 2.5. Cytotoxicity assay The effect of various DNA bound nanoparticles on cell viability was tested by trypan blue exclusion assay. An aliquot of cells was stained with 4% trypan blue vital dye and the blue stained dead cells were counted using a hemocytometer. 2.6. Polymerase chain reaction The pcDNA-GFP functionalized nanoparticles were subjected to PCR amplification using specific primers for pcDNA with 94 °C for 2 min to denature, 58 °C for 30 s for annealing, 70 °C for 1 min (40 cycles) for synthesis and final extension at 72 °C for 10 min. using MJ thermo cycler and the products were analyzed on 1% agarose gel. 3. Results and discussion Silver nanoparticles were synthesized using Mt, Ag + Mt, Ag + starch, Ag + citrate, Ag + polylysine and MWCNT as reported earlier (Rivas et al., 2001; Zhu Shiguo et al., 2002; Raveendran et al., 2003; Kiruba Daniel et al., 2011).
3.1. UV–visible absorption spectra The UV–visible absorption spectra of synthesized nanoparticles before functionalization with pcDNA-GFU showed a typical peak at 420 nm corresponding to the characteristic surface plasmon resonance of silver nanoparticles (Supplementary data) revealing that all the silver nanoparticle preparations showed a particle size of 20–25 nm as observed in earlier studies (Kiruba Daniel et al., 2010; Kiruba Daniel et al., 2011). 3.2. DNA binding The UV–visible absorption spectrum of DNA (Supplementary data) exhibited a broad band (200–350 nm) in the UV region with the maximum placed at 260 nm. In addition, the interband absorption at short wavelengths (except Mt and Ag-starch) increased, when a nanoparticle–DNA interaction existed. In the case of weaker interactions, only hypo chromic or hyper chromic effects are observed without significant changes of shifts in the spectral profiles. The amount of DNA adsorbed was more when the DNA was dissolved in distilled water for all silver nanoparticle preparations except for sodium citrate. 2-fold greater amounts of DNA were adsorbed only in the presence of excess divalent sodium (5 mM). DNA molecules are net negatively-charged and they can adsorb to net positively-charged surfaces, such as the edges of Mt (Nath et al., 2007), by electrostatic bridges with the water of hydration of chargecompensating cations (Paul et al., 2010). The aldehyde terminal of soluble starch was used to reduce silver nitrate while the extensive number of hydroxyl groups present in soluble starch facilitated the complexion of silver ions to the molecular matrix (Pinnavaia and Beall, 2000). Migration of a hydrogen atom within citric acid activates the electrons of the carboxyl oxygen and provides additional binding affinity toward silver (Rivas et al., 2001). The lysine residues in polylysine contain an epsilon amino group that forms an ionic interaction with DNA at high salt concentrations (Read et al., 1999; Shamsi and Geckeler, 2008). Cationic carbon nanotubes (CNT) are able to condense DNA to varying degrees, which determines the interaction and electrostatic complex formation between f-CNT with DNA. Upon the addition of divalent metal ions super coiled plasmid DNA forms relatively stable complexes with CNT due to chelation. 3.3. Fluorescence spectra of DNA functionalized silver nanoparticles In the presence of DNA, the metal nanoparticles showed an enhancement in the fluorescence polarization. This is due to the fact that the torsion vibrations and rotational motions are restricted. The photoluminescence spectra of the silver nanoparticles produced a typical emission peak at 420 nm and 553 nm when excited at 380 nm (Fig. 1). Similar results were observed even for MWCNT and Mt in the present study. The carboxylic acid group present in the MWCNT is readily available in anionic form like the DNA as observed by Suh and Chaires (1995) for MWCNT from mustard soot in water when excited at 364 nm. 3.4. Circular dichroism spectra The CD spectra (Fig. 2a) of the plasmid DNA functionalized nanoparticles revealed that the DNA was in B-form, as evidenced by a negative band at 248 nm, a slight red shift of the positive band at 280 nm and a considerable absorption in the longer wavelength (N 300 nm) indicating some perturbation of the DNA structure. The broadening of the negative band of DNA at 248 nm and other related changes had also been observed by Zherenkova et al. (2007) for the DNA-linked gold nanoparticle assemblies. In the case of gold nanoparticles, 2 nm sized nanoparticles can localize in the major groove of B-DNA. Gold nanoparticles
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Fig. 1. (A) The fluorescence spectrum (400–500 nm) of control and plasmid DNA functionalized various nanopreparations. 1. Mt, 2. Ag + Mt, 3. Ag + starch, 4. Ag + citrate, 5. Ag + polylysine, 6. MWCNT. (B) The fluorescence spectrum (500–600 nm) of control and plasmid DNA functionalized various nanopreparations. 1. Mt, 2. Ag + Mt, 3. Ag + starch, 4. Ag + citrate, 5. Ag + polylysine, 6. MWCNT.
interact with negatively charged oxygen atoms in phosphate groups and with pairs of nitrogen bases in DNA and thereby may disturb the electron structure and conformational homogeneity of nitrogen bases (chromophores) along the chain of the DNA molecule, which will lead to disturbance of the secondary structure of B-DNA (Zherenkova et al., 2007). Super coiled plasmid DNA interacts with a low number of strongly acidic groups, presumably located at the maximum of bending of the double strand where a high charge density exists. The Ag complexation did not alter DNA conformation (Arakawa et al., 2001).
study of Ag and pcDNA–GFU at a molar ratio of 1/5 revealed that Ag binds to guanine but not to adenine, thymine, cytosine, and the backbone phosphate groups (DiRico et al., 1985). DNA vibrational frequencies showed no major spectral changes upon silver cation interaction. A comparison of DNA binding to various nanoparticles (Fig. 3) showed that the DNA binding was more in the case of Ag + Mt and Ag + starch with a maximum binding with Ag + polylysine as cationic poly-lysine interacts with DNA cooperatively. 3.6. Polymerase chain reaction
3.5. Fourier transform IR pattern Fourier transform IR (FTIR) has been used to determine DNA binding sites and sequence preference, as well as conformational changes due to metal–DNA interaction (Jangir et al., 2010; Mandeville et al., 2010). Approximate position of IR bands of DNA and aqueous solvents were a) 1800–1500 cm− 1 region, sensitive to effects of base pairing and base stacking, b) 1500–1250 cm− 1 region, sensitive to glycosidic bond rotation, backbone conformation and sugar pucker, c) 1250–1000 cm−1 region, sensitive to backbone conformation, d) 1000–800 cm− 1 region, sensitive to sugar conformation (Fig. 2b). FTIR of DNA functionalized MWNTs showed a broad band at 3400 cm−1 which is attributed to the presence of O–H groups on the surface. The peak at 1720 cm− 1 is attributed to the C_O stretch of the carboxylic (COOH) group. The IR spectrum of the amidefunctionalized MWCNT samples, showed the disappearance of the band at 1720 cm− 1 and a corresponding appearance of a band with lower frequency (1661 cm−1) assigned to the amide carbonyl (C_O) stretch. Transition and non transition metal ions bind DNA through the backbone phosphate group at low cation concentration, whereas base binding occurs as metal ion concentration increases. The Ag(I) shows strong interaction for base binding with no affinity toward the backbone phosphate group at low or high cation concentration (Dattagupta and Crothers, 1981; DiRico et al., 1985). Based on these reports, it can be speculated that type I complexation with DNA involves not only G–C base pairs but also A–T bases. On the other hand, IR spectroscopic
The nanoparticles functionalized with plasmid DNA were subjected to polymerase chain reaction in order to verify the stability. A 5.1 kb amplicon was observed with the same size as bound DNA (Fig. 4). 3.7. In vitro transfection studies Transfection efficiency of various nanoparticles was assessed on liver cell line in vitro, using the plasmid pcDNA-GFP. Fig. 3 confirmed the efficient uptake of nanoparticles and efficient expression in all the preparations. The transfection efficiency was analyzed by measuring the fluorescence intensity of the GFP reporter attached to the pcDNA which showed the highest in vitro transfection efficiency (Fig. 5). The cell viability assay was performed on these cells after 4 days, wherein it was observed that cells treated with the nanoparticles showed significant increase in cell viability (≈98%) compared to 90% in the case of MWCNT and Ag + polylysine coated binding. Polylysine is a weak poly base. The interaction with DNA will be unfavorable since a greater proportion of the amino groups will be in the deprotonated state. At the nano-bio interface, internalization is mainly driven by electrostatic forces involving negatively charged proteoglycans and positively charged nanoparticles (Morille et al., 2008; Moreira et al, 2009). The dynamic molecular interactions and forces such as Coulombic electrostatic and steric attractive, van der Waals forces are established in the cell–particle interface. Therefore the larger effective positive surface charge obtained for particles with higher ratios accounts for their improved internalization capacity.
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Fig. 2. a. Circular dichroism pattern of functionalized nanoparticles. b. The FTIR pattern of functionalized nanoparticle. 1. Mt, 2. Ag + Mt, 3. Ag + starch, 4. Ag + citrate, 5. Ag + polylysine, 6. MWCNT.
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Fig. 3. Bar diagram showing the comparative plasmid DNA binding with various nanoparticles. (A) and (B) 1. Mt, 2. Ag + Mt, 3. Ag + starch, 4. Ag + citrate, 5. Ag + polylysine, 6. MWCNT (concentration calculated per 50 μg plasmid DNA). (C) and (D) 1. Mt, 2. Ag + Mt, 3. Ag + starch, 4. Ag + citrate, 5. Ag + polylysine.
The results indicated that although primary amine groups contribute significantly to transfection in the case of Ag + polylysine, they might have also disrupted the cellular integrity of the cell membrane due to an excessive positive charge on the nanoparticles (Shamsi and Geckeler, 2008). Positively charged nanocarriers disrupted the cell membrane causing death more than the anionic nanocarriers (Goodman et al., 2004, 2006; Osaka et al., 2009). Differences in the levels of gene expression (Fig. 6) were correlated with the structural and biophysical data obtained for the various products including MWCNT–DNA complexes to suggest that large surface area leading to very efficient DNA condensation is not necessary for effective gene transfer. Data on fluorescence pattern of DNA binding has other applications as well. Recently, several applications of SPR-based techniques have proven that it is possible to detect the presence of specific gene sequences without the need for PCR amplification or labeling of the sample since a single DNA molecule will increase the mass on the sensor surface much more than a drug molecule. The optical properties brought about by aggregation and network formation can be used as a
Fig. 4. PCR product of functionalized nanopreparations 1. Mt, 2. Ag + Mt, 3. Ag + starch, 4. Ag + citrate, 5. Ag + polylysine, 6. MWCNT, 7 & 8. Functionalized Ag + starch without PCR amplification (different concentrations). 9. 1 kb ladder (amplicon size is 5.1 kb).
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Fig. 6. Comparison of the transfection efficiency of various nanoparticles. 1. Mt., 2. Ag + Mt, 3. Ag + starch, 4. Ag + citrate, 5. Ag + polylysine, 6. MWCNT.
gratefully acknowledged. This study was carried out without any financial support.
References
Fig. 5. Transfection of liver cells with Ag-starch functionalized with pcDNA-GFP. 1. Cells under phase contrast microscope. 2. Cells under fluorescence microscope.
tool in DNA-detection including the diagnosis of genetic diseases, RNA profiling, biodefense (Hill et al., 2000; Kushon et al., 2003), gene chips (Lipshutz et al., 1999), detection of UV damage (Jiang et al., 2007) and single-molecule sequencing (Austin et al, 1997) including their use in nanopores (Gerland et al., 2004; Branton et al., 2008). The current problems associated with viral vectors as gene delivery systems are safety, immunogenicity and mutagenesis and also inefficient endosomal release, cytoplasmic transport and nuclear entry with toxicity. Results show that Ag + starch and Ag + Mt could be a cost effective nontoxic promising gene delivery system. Moreover, the newly developed multifunctional nanoparticles may potentially revolutionize the area of sensing, imaging, diagnosis and therapy. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.clay.2014.11.004.
Acknowledgment The authors thank the UPE and NET working facilities at Madurai Kamaraj University, Madurai for the use of their Fluorescence Spectrophotometer and Fluorescence Microscope. The FTIR and CD instruments spared by the School of Chemistry, Madurai Kamaraj University are
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Contents lists available at ScienceDirect
Applied Clay Science journal homepage: www.elsevier.com/locate/clay
Note
Comparison of nanocarriers for gene delivery and nanosensing using montmorillonite, silver nanoparticles and multiwalled carbon nanotubes T. Anitha Sironmani ⁎ School of Biotechnology, Madurai Kamaraj University, Madurai 625021, India
a r t i c l e
i n f o
Article history: Received 8 March 2014 Received in revised form 4 November 2014 Accepted 5 November 2014 Available online xxxx Keywords: DNA functionalizing Silver nanoparticles montmorillonite MWCNT SPR based DNA sensor Nonviral gene vector
a b s t r a c t Various types of nanoparticles like montmorillonite, silver nanoparticles stabilized with montmorillonite, starch, citrate, polylysine and multiwalled carbon nanotubes were used for binding with plasmid pcDNA-GFP in order to develop a gene delivery vector. The characterization of the functionalized materials showed a shift in the plasmon resonance to a higher wavelength compared to the control after DNA binding. This revealed the possibility that the DNA can be detected as a change in the absorption probability and/or a change in the resonance wavelength which can be used for diagnostic purposes. Transfection studies of these various functionalized nanopreparations implied that the gene delivery vector based on silver nanoparticles stabilized with starch and montmorillonite were more promising than other gene delivery systems with the potential to revolutionize the area of biosensing, imaging, diagnosis and therapy. © 2014 Elsevier B.V. All rights reserved.
1. Introduction For therapeutics, vaccination, replacing defective genes, gene delivery, abnormal cell destruction and for tissue regeneration, gene delivery vectors with minimal toxicity offer great potentials (O'Connor and Crystal, 2006; Wang and Yuan, 2006). The immune response elicited by viral proteins poses a major problem, leading to the development of nonviral DNA delivery vehicles. Nanoscience is an extremely powerful emerging science, which is expected to have a substantial impact on medical science now and in the future. The overall efficacy of nanoparticles to act as potential non viral gene delivery vehicles depends on the physical properties of nanoparticles, including their morphology, size, charge density and stability (Glover et al., 2005; Jackson et al., 2006; Khalil et al., 2006). Recently, synthesis of silver nanoparticles has attracted considerable attention owing to their diverse properties like catalysis (Shiraishi and Toshima, 2000), magnetic and optical polarizability (Shiraishi and Toshima, 2000), biosensor (Nimrodh Ananth et al., 2011), electrical conductivity (Chang and Yen, 1995), antimicrobial activity (Sharverdi et al., 2007) and Surface Enhanced Raman Scattering (SERS) (Matejka et al., 1992). In recent years, emerging applications of carbon nanotubes have shown their promising potentials in biomedical fields including novel delivery systems for drugs or DNA/RNA (Beg et al., 2011). Nanosilver can be modified for better efficiency to facilitate its diverse applications in medicine and life sciences. In order to enhance the biocompatibility and cell affinity for gene delivery, various methods of nanosilver preparation to functionalize with plasmid DNA for gene ⁎ Tel.: +91 0452 2458905. E-mail addresses: [email protected], [email protected].
http://dx.doi.org/10.1016/j.clay.2014.11.004 0169-1317/© 2014 Elsevier B.V. All rights reserved.
delivery were attempted. The green materials used in this study included montmorillonite (Mt), silver nanoparticles stabilized in montmorillonite (Ag + Mt), silver nanoparticles synthesized and stabilized with starch (Ag + starch), citrate (Ag + citrate) and polylysine (Ag + polylysine) and for comparison Mt and multiwalled carbon nanotube (MWCNT) were also used. The plasmid DNA (pcDNA) vector conjugated with complementary DNA of green fluorescent protein as reporter gene was used as the model DNA/gene to compare the performance of nanoparticles as gene delivery vectors. Understanding the unique characteristics of engineered nanomaterials and their interactions with DNA and biological systems is key to the safe implementation of these materials in novel biomedical diagnostics and therapeutics. Clay minerals have interesting chemical and physical characteristics, e.g., Mt has a high modulus, high cation exchange capacity, a large surface area to mass ratio and the ability to form stable dispersions in aqueous solutions (Marzilli, 1977; Bergaya et al., 2006). Clay mineral has been used successfully as vectors for delivery of DNA into cells in recent experiments (Zhu et al., 2002; Kiruba Daniel et al., 2010). The current study paves the base line on the biocompatible methods of synthesis of silver nanoparticles that enhances their usage in medicine for diagnostics and therapeutics. 2. Methods 2.1. Synthesis of nanoparticles The silver nanoparticles were synthetized in the interlamellar space of a layered Mt (Kiruba Daniel et al., 2010). Ag + starch was prepared as
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described in earlier studies (Kiruba Daniel et al., 2011). Ag + citrate and Ag + polylysine were prepared by following the earlier methods (Rivas et al., 2001; Zhu et al., 2002). Activated MWCNT prepared by the method developed by Sun et al. (2002) were provided as a gift by Prof. Saraswathy, Material Sciences, School of Chemistry, M.K. University, Madurai, Tamil Nadu, India. 2.2. Preparation of nanoparticle–DNA complexes A commercial solution of plasmid DNA, a complementary DNA for green fluorescent protein (GFP) containing vector (pcDNA-GFP vector, Clontech, Palo Alto, CA) was functionalized to various nanopreparations at a concentration of 50 μg ml−1 at 37 °C for 2 h. The nanoparticles and DNA were mixed for 2 h with and without salt. The functionalized nanoparticles were centrifuged at 10,000 rpm for a minute and the DNA content of the supernatants was measured at A260. The amount of adsorbed DNA was calculated by subtracting the amount present in the supernatant from the amount initially added. 2.3. Characterization of nanoparticles UV–visible spectra were recorded on a Shimadzu UV 1700. All the fluorescence data were obtained on a Perkin-Elmer LS50B luminescence spectrometer, The excitation wavelength used was 380 nm. CD spectra of control and DNA functionalized nanoparticles were taken in aqueous solutions at pH 7 under normal temperature using a JASCO J-810 Spectropolarimeter. FTIR spectra of all the functionalized nanoformulations were recorded on a Bruker Fourier transform infrared spectrometer (Vector 22 model) in the range 400–4000 cm−1 with a resolution of 4 cm−1. 2.4. Transfection efficiency Liver cells (from Swiss mouse), secondary culture (after passage 10, established in the lab) were seeded in Petri plates at a density of 5000 cells per plate in antibiotic-free DMEM with 10% serum. The amount of nanoparticles with DNA equivalent to 0.5 μg was added to each plate after 24 h. The cells were then incubated at 37 °C, 5% CO2 for 4 days, after which the cells were observed under fluorescence microscope (Nikon Eclipse TE2000-U) at a wavelength of 490 nm. Transfection with calcium phosphate-DNA was done as positive control. All transfection experiments were performed in triplicate. The transfection efficiency was calculated using the fluorescence expressed from the plasmid DNA encoding the GFP gene in pooled samples. 2.5. Cytotoxicity assay The effect of various DNA bound nanoparticles on cell viability was tested by trypan blue exclusion assay. An aliquot of cells was stained with 4% trypan blue vital dye and the blue stained dead cells were counted using a hemocytometer. 2.6. Polymerase chain reaction The pcDNA-GFP functionalized nanoparticles were subjected to PCR amplification using specific primers for pcDNA with 94 °C for 2 min to denature, 58 °C for 30 s for annealing, 70 °C for 1 min (40 cycles) for synthesis and final extension at 72 °C for 10 min. using MJ thermo cycler and the products were analyzed on 1% agarose gel. 3. Results and discussion Silver nanoparticles were synthesized using Mt, Ag + Mt, Ag + starch, Ag + citrate, Ag + polylysine and MWCNT as reported earlier (Rivas et al., 2001; Zhu Shiguo et al., 2002; Raveendran et al., 2003; Kiruba Daniel et al., 2011).
3.1. UV–visible absorption spectra The UV–visible absorption spectra of synthesized nanoparticles before functionalization with pcDNA-GFU showed a typical peak at 420 nm corresponding to the characteristic surface plasmon resonance of silver nanoparticles (Supplementary data) revealing that all the silver nanoparticle preparations showed a particle size of 20–25 nm as observed in earlier studies (Kiruba Daniel et al., 2010; Kiruba Daniel et al., 2011). 3.2. DNA binding The UV–visible absorption spectrum of DNA (Supplementary data) exhibited a broad band (200–350 nm) in the UV region with the maximum placed at 260 nm. In addition, the interband absorption at short wavelengths (except Mt and Ag-starch) increased, when a nanoparticle–DNA interaction existed. In the case of weaker interactions, only hypo chromic or hyper chromic effects are observed without significant changes of shifts in the spectral profiles. The amount of DNA adsorbed was more when the DNA was dissolved in distilled water for all silver nanoparticle preparations except for sodium citrate. 2-fold greater amounts of DNA were adsorbed only in the presence of excess divalent sodium (5 mM). DNA molecules are net negatively-charged and they can adsorb to net positively-charged surfaces, such as the edges of Mt (Nath et al., 2007), by electrostatic bridges with the water of hydration of chargecompensating cations (Paul et al., 2010). The aldehyde terminal of soluble starch was used to reduce silver nitrate while the extensive number of hydroxyl groups present in soluble starch facilitated the complexion of silver ions to the molecular matrix (Pinnavaia and Beall, 2000). Migration of a hydrogen atom within citric acid activates the electrons of the carboxyl oxygen and provides additional binding affinity toward silver (Rivas et al., 2001). The lysine residues in polylysine contain an epsilon amino group that forms an ionic interaction with DNA at high salt concentrations (Read et al., 1999; Shamsi and Geckeler, 2008). Cationic carbon nanotubes (CNT) are able to condense DNA to varying degrees, which determines the interaction and electrostatic complex formation between f-CNT with DNA. Upon the addition of divalent metal ions super coiled plasmid DNA forms relatively stable complexes with CNT due to chelation. 3.3. Fluorescence spectra of DNA functionalized silver nanoparticles In the presence of DNA, the metal nanoparticles showed an enhancement in the fluorescence polarization. This is due to the fact that the torsion vibrations and rotational motions are restricted. The photoluminescence spectra of the silver nanoparticles produced a typical emission peak at 420 nm and 553 nm when excited at 380 nm (Fig. 1). Similar results were observed even for MWCNT and Mt in the present study. The carboxylic acid group present in the MWCNT is readily available in anionic form like the DNA as observed by Suh and Chaires (1995) for MWCNT from mustard soot in water when excited at 364 nm. 3.4. Circular dichroism spectra The CD spectra (Fig. 2a) of the plasmid DNA functionalized nanoparticles revealed that the DNA was in B-form, as evidenced by a negative band at 248 nm, a slight red shift of the positive band at 280 nm and a considerable absorption in the longer wavelength (N 300 nm) indicating some perturbation of the DNA structure. The broadening of the negative band of DNA at 248 nm and other related changes had also been observed by Zherenkova et al. (2007) for the DNA-linked gold nanoparticle assemblies. In the case of gold nanoparticles, 2 nm sized nanoparticles can localize in the major groove of B-DNA. Gold nanoparticles
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Fig. 1. (A) The fluorescence spectrum (400–500 nm) of control and plasmid DNA functionalized various nanopreparations. 1. Mt, 2. Ag + Mt, 3. Ag + starch, 4. Ag + citrate, 5. Ag + polylysine, 6. MWCNT. (B) The fluorescence spectrum (500–600 nm) of control and plasmid DNA functionalized various nanopreparations. 1. Mt, 2. Ag + Mt, 3. Ag + starch, 4. Ag + citrate, 5. Ag + polylysine, 6. MWCNT.
interact with negatively charged oxygen atoms in phosphate groups and with pairs of nitrogen bases in DNA and thereby may disturb the electron structure and conformational homogeneity of nitrogen bases (chromophores) along the chain of the DNA molecule, which will lead to disturbance of the secondary structure of B-DNA (Zherenkova et al., 2007). Super coiled plasmid DNA interacts with a low number of strongly acidic groups, presumably located at the maximum of bending of the double strand where a high charge density exists. The Ag complexation did not alter DNA conformation (Arakawa et al., 2001).
study of Ag and pcDNA–GFU at a molar ratio of 1/5 revealed that Ag binds to guanine but not to adenine, thymine, cytosine, and the backbone phosphate groups (DiRico et al., 1985). DNA vibrational frequencies showed no major spectral changes upon silver cation interaction. A comparison of DNA binding to various nanoparticles (Fig. 3) showed that the DNA binding was more in the case of Ag + Mt and Ag + starch with a maximum binding with Ag + polylysine as cationic poly-lysine interacts with DNA cooperatively. 3.6. Polymerase chain reaction
3.5. Fourier transform IR pattern Fourier transform IR (FTIR) has been used to determine DNA binding sites and sequence preference, as well as conformational changes due to metal–DNA interaction (Jangir et al., 2010; Mandeville et al., 2010). Approximate position of IR bands of DNA and aqueous solvents were a) 1800–1500 cm− 1 region, sensitive to effects of base pairing and base stacking, b) 1500–1250 cm− 1 region, sensitive to glycosidic bond rotation, backbone conformation and sugar pucker, c) 1250–1000 cm−1 region, sensitive to backbone conformation, d) 1000–800 cm− 1 region, sensitive to sugar conformation (Fig. 2b). FTIR of DNA functionalized MWNTs showed a broad band at 3400 cm−1 which is attributed to the presence of O–H groups on the surface. The peak at 1720 cm− 1 is attributed to the C_O stretch of the carboxylic (COOH) group. The IR spectrum of the amidefunctionalized MWCNT samples, showed the disappearance of the band at 1720 cm− 1 and a corresponding appearance of a band with lower frequency (1661 cm−1) assigned to the amide carbonyl (C_O) stretch. Transition and non transition metal ions bind DNA through the backbone phosphate group at low cation concentration, whereas base binding occurs as metal ion concentration increases. The Ag(I) shows strong interaction for base binding with no affinity toward the backbone phosphate group at low or high cation concentration (Dattagupta and Crothers, 1981; DiRico et al., 1985). Based on these reports, it can be speculated that type I complexation with DNA involves not only G–C base pairs but also A–T bases. On the other hand, IR spectroscopic
The nanoparticles functionalized with plasmid DNA were subjected to polymerase chain reaction in order to verify the stability. A 5.1 kb amplicon was observed with the same size as bound DNA (Fig. 4). 3.7. In vitro transfection studies Transfection efficiency of various nanoparticles was assessed on liver cell line in vitro, using the plasmid pcDNA-GFP. Fig. 3 confirmed the efficient uptake of nanoparticles and efficient expression in all the preparations. The transfection efficiency was analyzed by measuring the fluorescence intensity of the GFP reporter attached to the pcDNA which showed the highest in vitro transfection efficiency (Fig. 5). The cell viability assay was performed on these cells after 4 days, wherein it was observed that cells treated with the nanoparticles showed significant increase in cell viability (≈98%) compared to 90% in the case of MWCNT and Ag + polylysine coated binding. Polylysine is a weak poly base. The interaction with DNA will be unfavorable since a greater proportion of the amino groups will be in the deprotonated state. At the nano-bio interface, internalization is mainly driven by electrostatic forces involving negatively charged proteoglycans and positively charged nanoparticles (Morille et al., 2008; Moreira et al, 2009). The dynamic molecular interactions and forces such as Coulombic electrostatic and steric attractive, van der Waals forces are established in the cell–particle interface. Therefore the larger effective positive surface charge obtained for particles with higher ratios accounts for their improved internalization capacity.
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Fig. 2. a. Circular dichroism pattern of functionalized nanoparticles. b. The FTIR pattern of functionalized nanoparticle. 1. Mt, 2. Ag + Mt, 3. Ag + starch, 4. Ag + citrate, 5. Ag + polylysine, 6. MWCNT.
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Fig. 3. Bar diagram showing the comparative plasmid DNA binding with various nanoparticles. (A) and (B) 1. Mt, 2. Ag + Mt, 3. Ag + starch, 4. Ag + citrate, 5. Ag + polylysine, 6. MWCNT (concentration calculated per 50 μg plasmid DNA). (C) and (D) 1. Mt, 2. Ag + Mt, 3. Ag + starch, 4. Ag + citrate, 5. Ag + polylysine.
The results indicated that although primary amine groups contribute significantly to transfection in the case of Ag + polylysine, they might have also disrupted the cellular integrity of the cell membrane due to an excessive positive charge on the nanoparticles (Shamsi and Geckeler, 2008). Positively charged nanocarriers disrupted the cell membrane causing death more than the anionic nanocarriers (Goodman et al., 2004, 2006; Osaka et al., 2009). Differences in the levels of gene expression (Fig. 6) were correlated with the structural and biophysical data obtained for the various products including MWCNT–DNA complexes to suggest that large surface area leading to very efficient DNA condensation is not necessary for effective gene transfer. Data on fluorescence pattern of DNA binding has other applications as well. Recently, several applications of SPR-based techniques have proven that it is possible to detect the presence of specific gene sequences without the need for PCR amplification or labeling of the sample since a single DNA molecule will increase the mass on the sensor surface much more than a drug molecule. The optical properties brought about by aggregation and network formation can be used as a
Fig. 4. PCR product of functionalized nanopreparations 1. Mt, 2. Ag + Mt, 3. Ag + starch, 4. Ag + citrate, 5. Ag + polylysine, 6. MWCNT, 7 & 8. Functionalized Ag + starch without PCR amplification (different concentrations). 9. 1 kb ladder (amplicon size is 5.1 kb).
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Fig. 6. Comparison of the transfection efficiency of various nanoparticles. 1. Mt., 2. Ag + Mt, 3. Ag + starch, 4. Ag + citrate, 5. Ag + polylysine, 6. MWCNT.
gratefully acknowledged. This study was carried out without any financial support.
References
Fig. 5. Transfection of liver cells with Ag-starch functionalized with pcDNA-GFP. 1. Cells under phase contrast microscope. 2. Cells under fluorescence microscope.
tool in DNA-detection including the diagnosis of genetic diseases, RNA profiling, biodefense (Hill et al., 2000; Kushon et al., 2003), gene chips (Lipshutz et al., 1999), detection of UV damage (Jiang et al., 2007) and single-molecule sequencing (Austin et al, 1997) including their use in nanopores (Gerland et al., 2004; Branton et al., 2008). The current problems associated with viral vectors as gene delivery systems are safety, immunogenicity and mutagenesis and also inefficient endosomal release, cytoplasmic transport and nuclear entry with toxicity. Results show that Ag + starch and Ag + Mt could be a cost effective nontoxic promising gene delivery system. Moreover, the newly developed multifunctional nanoparticles may potentially revolutionize the area of sensing, imaging, diagnosis and therapy. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.clay.2014.11.004.
Acknowledgment The authors thank the UPE and NET working facilities at Madurai Kamaraj University, Madurai for the use of their Fluorescence Spectrophotometer and Fluorescence Microscope. The FTIR and CD instruments spared by the School of Chemistry, Madurai Kamaraj University are
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