A supramolecular hydrogel for generation of a benign DNA-hydrogel

Abstract

A novel hydrogel has been synthesized using 2′,4′,6′-tri(4-pyridyl)pyridine in an acidic water medium. TEM analyses along with rheological experiments explore the supramolecular features of the hydrogel. In vitro and in vivo toxicity studies depict the non-toxic and bio-relevant nature of the pure supramolecular hydrogel and its aqueous solution. This bio-compatible supramolecular hydrogel is used for the synthesis of a DNA-hydrogel with calf thymus DNA. The DNA-hydrogel is intriguing for the effective stabilization of photochemically synthesized silver nanoparticles from Ag(I) under the direct exposure of sunrays. DNA-hydrogel capped Ag-NPs are also bio-compatible. The luminous features of the 2′,4′,6′-tri(4-pyridyl)pyridine based supramolecular hydrogel, DNA-hydrogel and DNA-hydrogel capped Ag-NPs and their aqueous solutions are explored through fluorescence microscopy and spectral analyses, respectively.

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1. Introduction

Hydrogels are chemically engineered three-dimensional polymers with excessively trapped water molecules.^1,2 A hydrogel is intriguing not only due to its bio-functionalities for tissue engineering,³ as a cell-support,^4,5 and for drug delivery,⁶ but also for miscellaneous material applications encompassing chemo-sensing,⁷ photolithography,⁸ microfluidic designing⁹ and electrochemical tasks.¹⁰ A luminescent hydrogel matrix is also used for bio-signaling purposes.¹¹ Oligopyridine chromophore based luminous hydrogel synthesis^12,13 is a novel technique to develop soft functional materials. The DNA-intercalation feature of protonated pyridine based small molecules¹⁴ along with the affinity of the negatively charged phosphate backbone in DNA with positively charged protonated oligopyridines are utilized to synthesize the DNA-hydrogel.^15–18 DNA-hydrogels have various functionalities including mechanical features,¹⁵ bio-molecule detection,¹⁶ nanoparticle stabilization,^17,18 etc.

In this work we have developed a protonated 2′,4′,6′-tri(4-pyridyl)pyridine based supramolecular hydrogel network. This hydrogel also produces a DNA-hydrogel with calf thymus DNA. Following the literature¹⁹ we have attempted to photochemically synthesize silver nanoparticles in a DNA based benign hydrogel medium. The DNA-hydrogel can effectively stabilize the photochemically generated silver nanoparticles from Ag(I) under the direct exposure of sunrays. The supramolecular hydrogel, DNA-hydrogel and DNA-hydrogel capped Ag-NPs are luminescent. In addition, toxicity of the hydrogel and Ag-NPs are also investigated through in vitro and in vivo studies using the MTT assay and Wistar rat (Rattus norvegicus) model, respectively.

2. Experimental section

2.1 Materials

All chemicals and related consumables have been purchased from Sigma-Aldrich, Merck, Thermo scientific, and NEST biotechnology and used as received. Nuclease free type 18 Milli-Q water is used throughout. Calf thymus DNA (i.e. CT-DNA), purchased from Merck, has been used as the source of DNA throughout the study.

2.2 Characterization

Absorption and fluorescence spectra were measured using a SHIMADZU UV-1800 and SHIMADZU UV-3101PC spectrophotometer and a Perkin-Elmer LS55 fluorescence spectrometer, respectively. The circular dichroism spectroscopy study was performed using an Applied Photophysics Chirascan CD with a detection range of 160–850 nm. Elemental analysis (C, H, and N) was carried out using a Perkin-Elmer 240C elemental analyzer. Electrospray ionisation mass spectroscopy (ESI-MS) experiments were carried out on a Water’s QtoF Model YA 263 spectrometer in the positive ion ESI mode. The pK_a value was measured using a Metrohm 888 Titrando made autotitrator. The TEM images were taken with a FEI Tecnai G² F30 S-Twin microscope using an accelerating voltage of 300 kV. The TEM apparatus is also equipped with a GATAN Orius CCD camera, and a high-angle annular dark field Scanning Transmission Electron Microscopy (STEM-HAADF) mode with a HAADF detector from Fischione (model 3000). The compositional analysis was performed using an energy dispersive X-ray spectroscopy (EDS, EDAX Inc.) attachment on the Tecnai G² F30. Energy-filtered TEM (EFTEM) measurements were carried out using a Gatan Imaging Filter Quantum SE (model 963). The samples were dispersed in deionized water by sonication and dropped onto a conventional carbon-coated copper grid. SEM images were obtained using a Scanning Electron Microscopy Hitachi S-530 model with a sputter Gold-coater and critical point dryer. The rheological experiment was carried out using an Anton Paar made RHEOPLUS/32 V3.40 rheometer and a rheometer (TA Instrument) using cone plate geometry. The dynamic light scattering (DLS) study was performed using a Malvern instrument. In situ fluorescence studies for Shgel were imaged using a gel-documentation system with UV transillumination made by Bio-Rad Laboratories, USA. The UV microscopy study was performed using a Leica 224 Fluorescence Microscope with a UV attachment. Fluorescence microscopy studies were performed using an Inverted Fluorescence Microscope, made by Victory-Dewinter, Italy. Cell cultures were conducted in a humidified CO₂ incubator made by New Brunswick, Eppendorf, Germany. Fluorescence lifetimes were measured using a HORIBA JOBIN Yvon single photon counting setup and a nano LED with a 370 nm light source for excitation. ¹H and ¹³C NMR spectra were measured using a Bruker 400 MHz instrument.

2.3 Ethical clearance

Animal related studies were approved by Institutional Ethical Committee for Animal and Human, Visva-Bharati, India.

2.4 Synthesis of the luminescent supramolecular hydrogel (Shgel)

2′,4′,6′-Tri(4-pyridyl)pyridine (i.e. tetrapyridine) was synthesized according to the literature method²⁰ (C, H, and N analysis data for tetrapyridine: anal. calcd for tetrapyridine, C₂₀H₁₄N₄: C, 77.42; H, 4.51; N, 18.06%. Found: C, 77.39; H, 4.50; N, 18.02%. ¹H NMR (CDCl₃, ppm) δ: 8.819 (m, 6H_Py), 8.083 (m, 6H_Py), 7.654 (d, 2H_Py, J = 6 Hz); ¹³C NMR (CDCl₃, ppm) δ: 156.02, 151.06, 150.81, 145.71, 145.56, 121.72, 121.33, 118.86. See ¹H and ¹³C NMR spectra in ESI as Fig. S1†). This tetrapyridine compound (1 mM, 0.310 g) was dissolved in a 20 ml water solution of nitric acid (H₂O : HNO₃ = 1 : 1 v/v) at room temperature (∼25.0 °C). Then the reaction mixture was continuously stirred for 30 minutes and kept for gelation at room temperature under atmospheric pressure. A white coloured hydrogel was obtained after eleven days at room temperature (see ESI for Scheme S1†). This hydrogel is also stable up to seven months at room temperature under atmospheric pressure. The inherently adjusted program of the autotitrator shows that there are two different pK_a values (i.e. pK_a1 = 2.17 ± 0.05, and pK_a2 = 5.54 ± 0.10) for the pure tetrapyridine ligand in the water medium and this has been possibly rationalized due to the presence of two different types of nitrogen atoms from the peripheral tripyridyl rings and the central pyridine ring of the pure tetrapyridine ligand. This hydrogel was collected for characterization and further progress of our presented work (see ESI† for ESI-MS patterns of the pure tetrapyridine ligand and Shgel given as Fig. S2).† According to the literature²¹ the ‘T_Gel’ (i.e. gel melting temperature at different concentrations of the gelator) of the Shgel is shown in ESI as Fig. S3.† The dynamic light scattering study of the Shgel is also given as Fig. S4† in ESI.†

2.5 Synthesis of the DNA-hydrogel

For the preparation of the DNA-hydrogel, a stock aqueous solution of the Shgel with pH of ∼4.71 ± 0.10 having a concentration of 100 μg ml⁻¹ (Shgel/water) was taken. The mixture of a 100 μl ([CT-DNA] = 100 μg ml⁻¹) water solution of CT-DNA and a 900 μl ([Shgel] = 100 μg ml⁻¹) water solution of the luminescent Shgel in a test tube immediately leads to the formation of a DNA-hydrogel loaded at the reaction tube (see ESI for Scheme S2†).

2.6 Synthesis of the DNA-hydrogel capped Ag-NPs

The DNA-hydrogel (0.25 g) and silver nitrate (0.09 g) were thoroughly mixed in a watch glass. The mixture was kept under an open exposure of direct sunlight and instantaneously a violet coloured mass, also specifying the reduction of silver ions (Ag⁺) to silver nanoclusters (Ag⁰),²² was obtained. Finally, this violet coloured mass was examined for depicting the formation of silver nanoparticles (see ESI for Scheme S3†). The control experiment (i.e. using pure CT-DNA instead of the DNA-hydrogel) for the photochemical synthesis of Ag-NPs has also been performed (see ESI for the UV-VIS pattern of pure CT-DNA capped Ag-NPs as Fig. S5†).

2.7 In vitro cytotoxicity assessment for the aqueous solution of Shgel

For the in vitro assessment, a batch of aqueous solutions with dissimilar doses (viz. 50, 100 and 200 μg ml⁻¹ with a pH range ∼4.53–4.87 ± 0.10) of the Shgel were aseptically added into rat peritoneal macrophages (2 × 10⁶) plated in six well culture vessels which were incubated for 6 h and after incubation, 200 μl of a MTT solution (in 100 mM PBS) was added to each of the culture vessels with a further 1 hour incubation at 37 °C under a dark atmosphere. Finally, these treated cells were isolated by scraping and centrifuged at 4000 rpm for 2 min. 200 μl of DMSO was added into each treated solution containing cells and colour intensities were measured at 595 nm using a plate reader (Beckman, USA) for each case.

2.8 In vivo toxicity assessment of the Shgel

A chronic exposure of the direct Shgel (100 μg) was given to separate groups of adult male Wistar rats with one control group (n = 6; 120 ± 10 g) intraperitoneally, subcutaneously and gustatory for 30 days consecutively. All the rats were supplied with water and feed ad libitum with constant monitoring for any behavioural abnormalities and/or weight loss. Diverse serological and biochemical means were evaluated maintaining conventional standard protocols.

N. B.: due to the non-toxic nature of the Shgel, we have omitted the toxicity study of the DNA-hydrogel, as Shgel is the elementary unit of the DNA-hydrogel.

2.9 In vitro cytotoxicity assessment of the Ag-NPs

Aqueous solutions with dissimilar doses (viz. 2.5, 5 and 10 μg ml⁻¹) of the DNA-hydrogel capped Ag-NPs were tested following a similar method as mentioned for the Shgel.

2.10 In vivo toxicity assessment of the Ag-NPs

In order to judge the toxicity of Ag-NPs inside living entities, the Wistar rat (Rattus norvegicus) model has been implemented. A chronic exposure of Ag-NPs (100 μg) were given to separate groups of adult male Wistar rats following a similar procedure as applied for the Shgel.

2.11 Study of the DNA-interaction by a fluorescence spectroscopy method

Batches of water solutions of CT-DNA with increasing concentrations have been successively added into the water solution of the Shgel (i.e. [Shgel] = 100 μg ml⁻¹) and thereby corresponding fluorescent signals of the water solutions containing the Shgel and CT-DNA have also been recorded. The concentrations of CT-DNA used for the fluorescence spectroscopy study are 0, 0.107 × 10⁻⁷, 0.115 × 10⁻⁶, 0.122 × 10⁻⁵, 0.13 × 10⁻⁴, and 0.137 × 10⁻³ g ml⁻¹.

2.12 Measurements of average excited state lifetimes (τ_avg) of the aqueous Shgel solution in the absence and in the presence of CT-DNA

Time resolved fluorescence decay of the aqueous Shgel solution (with a concentration of 1 μg ml⁻¹) in the absence and presence of added CT-DNA (with a concentration of 10⁻⁴ μg ml⁻¹) has been measured in a water medium at 25 °C using a nano LED of 370 nm as the light source at λ_em = 562 nm. Here, the χ² values for the aqueous Shgel solution in the absence and presence of CT-DNA are 1.004964 and 1.006092, respectively. The average excited state lifetimes (τ_avg) of the aqueous Shgel solution in the absence and presence of CT-DNA are 3.01 and 3.02 nanoseconds, respectively.

2.13 Circular dichroism spectroscopy study of the aqueous Shgel solution with CT-DNA

The water solution of CT-DNA with a concentration of 1 μg ml⁻¹ was used for this study. The instrument parameters were set at a scanning speed of 50 nm min⁻¹, bandwidth of 10 nm and sensitivity of 100 millidegrees. Four scans were averaged and smoothed to improve the signal to noise ratio. The molar ellipticity values are expressed in terms of the mean residue molar ellipticity (in deg cm² dmol⁻¹). Secondary structure analysis was performed by the software supplied by Applied Photophysics Chirascan CD (see also ESI† for CD spectral analysis of the Ag-NPs and CT-DNA).

2.14 Gel-electrophoresis study

Gel electrophoresis was done in a 1.5% agarose gel slab submerged in a tris–EDTA boric acid buffer (pH = 7.0) loaded in the submarine of the gel-electrophoresis setup and supplied with a constant power of 100 V for one hour. Upon completion of electrophoresis, the gel was visualized and imaged under UV transillumination (only DNA containing samples were post-stained with 1 mg ml⁻¹ ethidium bromide in the above-mentioned buffer solution).

3. Results and discussion

3.1 Microstructural analysis of the 2′,4′,6′-tri(4-pyridyl)pyridine based Shgel

The structure and chemistry of the Shgel were visualized using transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDS) and energy-filtered TEM (EFTEM) techniques (Fig. 1). TEM images showed a thin plate-like morphology of the Shgel (Fig. 1). A higher magnification image (Fig. 1a) further illustrates that the hydrogel is indeed a collection of acute thin plates. The selected area electron diffraction (SAED) patterns, taken from different regions, show diffuse rings. One such SAED pattern shown in the inset of Fig. 1c undoubtedly shows the characteristics of an amorphous material. The EDX spectra were collected from different regions of the Shgel using a high-angle annular dark field scanning/transmission electron microscopy (HAADF-STEM) mode to get elemental information. The HAADF image is shown in Fig. 1d, whereas Fig. 1e exhibits the corresponding representative EDX spectrum from this sample and the spectrum reveals the presence of C, O, and Cl. The Cu signal is due to a grid and the C signal is a combination of a carbon coated grid and the Shgel, which was confirmed further using EFTEM imaging (Fig. 1f). There is a prominent π⋯π stacking interaction within the Shgel network which is evident from the stacked plate-like pattern observed in the TEM analysis (Fig. 1a). Due to the protonation in the ring nitrogen atoms, tetrapyridine ring hydrophobicity of the Shgel is lowered compared to that of its non-protonated state. The lowered interfacial energy in a polar arena of water, achieved through the π⋯π stacked protonated tetrapyridine rings at the inner core and the associated hydrophilic nitrate anions in its peripheral zones, along with possible non-covalent type interactions, are probably accountable for the generation of the luminescent supramolecular nano-scale hydrogel pattern of the Shgel.¹³

Fig. 1 (a) TEM images (I and II) taken at two magnifications represent a thin sheet of layers confirming a nano-scale supramolecular structure. Magnified TEM images (III and IV) of the regions marked by dotted squares 1 and 2 in (a) (II), respectively, clearly show the supramolecular architecture. (b) TEM image of the hydrogel sample taken at low magnification. (c) Magnified image of the region marked by a red coloured dotted square in (b) and in the inset is the SAED pattern. (d) STEM-HAADF image of the hydrogel. (e) EDX spectrum of the gel from a region marked by the red coloured area 1 in (d). (f) EFTEM images taken from the same red dotted square area in (b).

3.2 Rheological analysis of the Shgel

Supramolecular gels are viscoelastic materials and these always display a combination of viscous and elastic properties characterized by rheological experiments. Their behavior can lead to the accumulation and dissipation of energy under oscillatory stress and is characterized by parameters called the storage (G′) and loss (G′′) modulus of the sample, respectively. The storage modulus (G′) and loss modulus (G′′) of the samples are defined as:²³

G′ = (σ₀/γ₀)cos(δ)

G′′ = (σ₀/γ₀)sin(δ)

The storage modulus (G′) represents distortion of stored energy of the samples during the application of shearing on the viscoelastic region. The loss modulus (G′′) represents the way energy is dissipated from the system during the shearing process and shows the flow or liquid-like nature. In the gel state, it follows G′(ω) > G′′(ω) and G′(ω) ≈ ω°, where ω is the angular frequency. From the rheological data, the mechanical strength (G′), elasticity (G′ − G′′) and stiffness (G′/G′′) of the supramolecular gels are also measured. The frequency sweep experiment of the supramolecular gel (i.e. Shgel) with a 1% strain is shown in Fig. 2. Fig. 2a shows that G′ and G′′ follow a wide linearity in the viscoelastic region with an angular frequency and G′ value (16 895 Pa) that is considerably higher than that of G′′ (10 175 Pa). It confirms the hydrogel-network nature of the system and shows the solid-like nature of the sample (i.e. Shgel).²⁴ The Shgel exhibits a stress-resistant property up to a satisfactory range of oscillator stress at a constant frequency of 1 Hz (Fig. 2b) showing a yield stress (σ*) value of 100.5 Pa. The high yield stress may develop from a high stiffness (G′/G′′ = 1.7) of the hydrogel due to formation of a nano-fibrous morphology of this hydrogel system. The high surface area of the nano-fibrous nature, having more interaction sites, and the presence of very high compactness suggest that the Shgel is a tough material.²⁵ The rheological experiments show that the viscosity (η) of the Shgel falls in the range of 0.005–5 Pa s at a temperature range of 30–50 °C (see ESI for details, Fig. S6–S8†).

Fig. 2 (a) Angular frequency-dependent storage modulus (G′) and loss modulus (G′′) of the Shgel. (b) Plot of G′ and G′′ vs. oscillator stress at a constant frequency of 1 Hz at 30 °C.

3.3 Toxicity studies of the aqueous Shgel solution

Approaching the attainment of optimal bio-compatibilities, a toxicity assessment for the aqueous Shgel solution has been performed in vitro and in vivo. The in vitro assessment via a dose dependent MTT assay of the aqueous Shgel solution with rat peritoneal macrophages shows that the aqueous Shgel solution is non-toxic. Subsequently, the following Wistar rat (Rattus norvegicus) model in vivo toxicity analysis also shows that the water solution of Shgel is non-toxic and explicitly bio-compatible (Fig. 3 and see ESI for Table S1†). The bio-friendly nature of the aqueous Shgel solution led us to create another novel hydrogel pattern with a bio-macromolecule like CT-DNA.

Fig. 3 In vitro cytotoxicity assessment of the water solution of the Shgel: (a) MTT assay with 50, 100, and 200 μg ml⁻¹ of the aqueous Shgel solution depicting % viability of rat peritoneal macrophage cells. (b) Phase contrast microscopy images of the control and aqueous Shgel solution treated macrophages in culture.

3.4 Microstructural study of the DNA-hydrogel

The presence of supramolecular features and protonated tetrapyridinium moieties within the Shgel network was employed for the generation of a DNA-hydrogel pattern with CT-DNA. The direct mixing of a microgram level aqueous solution of CT-DNA and the Shgel produces an intriguing DNA-hydrogel network (Fig. 4). The microstructure of the DNA-hydrogel was also analysed by a TEM study (Fig. 4) which clearly shows a novel nano-scale hydrogel pattern of the DNA-hydrogel.

Fig. 4 TEM images showing the microstructure of the DNA-hydrogel where the red coloured circular marks indicate the presence of CT-DNA in DNA-hydrogel networks (see ESI for Fig. S9† comprising the SEM microstructure of the DNA-hydrogel).

3.5 Synthesis of the DNA-hydrogel capped Ag-NPs

Under the direct exposure of sunrays, silver nanoparticles have been synthesized from silver nitrate instantaneously in a DNA-hydrogel bed. TEM results (Fig. 5) along with the EDX spectrum (Fig. 5f) show that Ag-NPs are photochemically generated under sunray exposure and the DNA-hydrogel acts as an efficient capping agent of Ag-NPs. The DNA-hydrogel in its bloated juncture affords a large free space which might be responsible for the nucleation, precise growth and essential stabilization of Ag-NPs.²²

Fig. 5 TEM images of silver nanoparticles capped with a DNA-hydrogel are shown in (a–e) and the EDX spectrum, shown in (f), from a red region marked by 1 in (e), confirms the presence of Ag-NPs embedded in the DNA-hydrogel.

3.6 Toxicity analysis of the DNA-hydrogel capped Ag-NPs

The toxicity assessments for Ag-NPs are performed in vitro (via the MTT assay with rat peritoneal macrophages) and in vivo (following the Wistar rat model) (Fig. 6 and see ESI for Table S2†). In vitro and in vivo assessments confirm that these Ag-NPs are also extremely bio-compatible. CD spectroscopy analysis also shows that minor groove binding via non-covalent type interactions dictates the interaction features between Ag-NPs and CT-DNA (see ESI for Fig. S10†). Minor groove binding is revealed by the absence of characteristic changes except the intensity in the CD spectrum showing decoiling of the DNA double helix after exposure to Ag-NPs under ambient conditions. These experiments have also keenly motivated us towards the aim of using the DNA-hydrogel for in vivo synthesis applications of biologically benign metal nanoparticles.

Fig. 6 (A) Mortality evaluation of rat peritoneal macrophages in the presence of Ag-NPs using the MTT assay. (B) Bio-compatibility study: phase contrast microscopy images of control rat macrophages and macrophages treated with Ag-NPs. Here, rat peritoneum macrophages were treated with Ag-NPs separately with their above-mentioned highest doses and imaged under an inverted phase contrast microscope.

3.7 Fluorimetric DNA signaling by the water solution of the Shgel and luminous Ag-NPs

The interactions between CT-DNA and the water solution of the Shgel have also been explored through a spectroscopy technique. The aqueous solution of the Shgel shows a broad absorption band centred at 350 nm and a fluorescence emission maximum at 562 nm (λ_ex = 350 nm) at 298 K and under atmospheric pressure (Fig. 7 and see ESI for Fig. S11 and S12†). The fluorescent feature of the aqueous Shgel solution has been employed to directly explore the nature of the interaction between CT-DNA and the Shgel in a water medium. The fluorescence intensity of the water solution of the Shgel has been monotonically enhanced due to the addition of different aqueous solutions of CT-DNA at increasing concentrations ranging from the sub-microgram (i.e. 0.107 × 10⁻⁷ g ml⁻¹) to milligram level (i.e. 0.137 × 10⁻³ g ml⁻¹) into the aqueous solution of the Shgel (Fig. 7a). The fluorescence spectral data indicates that mechanistically non-covalent interactions operating between the negatively charged phosphate backbone of CT-DNA and the protonated tetrapyridine rings of the aqueous Shgel solution may be key accountable factors for this dose dependent monotonic fluorescence enhancement (Fig. 7a). The above mentioned mechanism of DNA-interaction might be justified by almost unchanged fluorescence average lifetimes of the aqueous solution of the pure Shgel (τ_avg = 3.01 ns) and Shgel with CT-DNA (τ_avg = 3.02 ns) found in the time resolved fluorescence decay measurements (inset of Fig. 7 as Fig. 7b). The luminescent property of the DNA-hydrogel has been confirmed by these fluorescence spectra (i.e. the spectral lines of the aqueous Shgel solution, placed above the initial black coloured line, found in the presence of CT-DNA with its various concentrations given as Fig. 7a) and the microscopy study (see ESI for Fig. S13†). TEM analysis (Fig. 4) shows that during the formation of the DNA-hydrogel, DNA strands are assembled with the luminescent Shgel network to form a novel organized gel-structure and this probably imposes the luminescent property into the resultant DNA-hydrogel material. The luminous nature of DNA-hydrogel capped Ag-NPs has also been investigated through fluorescence spectroscopy and microscopy studies (see ESI for Fig. S14†). Due to the luminescent property of the DNA-hydrogel, DNA-hydrogel capped Ag-NPs are also luminous. The dilute aqueous solution of DNA-hydrogel capped Ag-NPs shows a fluorescence emission maximum at 548 nm (λ_ex = 350 nm) at 298 K and under atmospheric pressure (see ESI for Fig. S14†) whereas the control experiment i.e. the fluorescence pattern of the water solution of pure CT-DNA capped Ag-NPs shows its non-luminous nature.

Fig. 7 (a) Fluorescent signaling of sub-microgram to milligram level CT-DNA by the aqueous Shgel solution. Fluorescence spectroscopy pattern of a water solution of the Shgel in the presence of different aqueous solutions of CT-DNA at varying concentrations (i.e. 0 to 0.137 × 10⁻³ g ml⁻¹). The initial black coloured spectrum denotes the fluorescence pattern of the pure Shgel in a water medium. (b) Time resolved fluorescence decay of the aqueous Shgel solution at a 1 μg ml⁻¹ concentration in the absence and presence of externally added CT-DNA given in the inset of this figure.

3.8 CD spectral analysis and gel-electrophoresis study

Due to the addition of the aqueous Shgel solution into an aqueous solution of CT-DNA, the intensity of the CD spectroscopy signal of DNA has been altered (Fig. 8a). CD spectral data (Fig. 8a) clearly shows that there is a non-covalent type groove (minor) binding interaction between the protonated tetrapyridine rings of the aqueous Shgel solution and CT-DNA.^26–30 The gel-electrophoresis study (Fig. 8b and c) also endorses the strong affinity of the luminous aqueous Shgel solution towards CT-DNA via exhibiting its retention features within the gel wells, whereas the absence of the aqueous Shgel solution completely reverses the phenomena i.e. cataphoresis of unbound DNA in its usual corridor (Fig. 8c).

Fig. 8 (a) CD spectroscopy result of pure CT-DNA in the absence and presence of the Shgel in a water medium. (b) (i) Dose dependent luminescent nature of the aqueous Shgel solution embedded within gel wells and (b) (ii) corresponding densitometric result. This densitometric result supports the dose dependent monotonic enhancement of the luminescent feature of gel wells filled with the aqueous Shgel solution. (c) Binding efficiency of the aqueous Shgel solution with CT-DNA found in gel-electrophoresis. Here, five wells and their related lanes from the left side show a strong binding affinity of CT-DNA towards the protonated tetrapyridine rings of the aqueous Shgel solution and simultaneously, the last three wells and their related lanes exhibit the binding features of ethidium bromide towards CT-DNA.

4. Conclusion

In summary, we have prepared a protonated tetrapyridinium moiety based hydrogel having a supramolecular network. Being a photoresponsive chromophore, 2′,4′,6′-tri(4-pyridyl)pyridine is the key constituent for achieving the luminescent hydrogel structure. TEM microstructural analysis along with a rheological study clearly show the supramolecular nano-scale pattern of the Shgel with high mechanical stability. In vitro and in vivo toxicity studies, via the MTT assay and Wistar rat model, respectively, show that the Shgel in a water solution is non-toxic. Owing to its non-toxicity, supramolecular features and the presence of positively charged protonated tetrapyridinium rings, the aqueous Shgel solution may form another hydrogel pattern with DNA, having a negatively charged phosphate backbone. This conjecture is materialized through the formation of a DNA-hydrogel with calf thymus DNA. Most intriguingly, under the direct exposure of sunlight, Ag-NPs were instantly photochemically synthesised and these Ag-NPs become efficiently stabilized in a DNA-hydrogel platform. In addition, in vitro and in vivo studies, via the MTT assay and Wistar rat model, respectively, confirm that these synthesized Ag-NPs are also bio-compatible. Through this work it has been justified that bio-compatible Ag-NPs can be photochemically synthesized under the exposure of sunrays where the benign DNA-hydrogel network acts as a capping agent for Ag-NPs. Fluorescence microscopy and spectral analysis confirm that the aqueous Shgel solution is luminous. TEM microstructural analysis of the Shgel and DNA-hydrogel shows that the Shgel forms a supramolecular network while DNA strands are spread into the Shgel network in the DNA-hydrogel. Due to the fluorescent nature of the aqueous Shgel solution, the DNA-hydrogel is also luminescent. Fluorescence spectroscopy analysis clearly indicates that there are non-covalent type supramolecular interactions between a wide range of CT-DNA (i.e. from the sub-microgram to milligram level) and the Shgel in a water medium. CD spectral analysis also ratifies that the aqueous Shgel solution can non-covalently interact with CT-DNA through its minor groove binding in a water medium. A gel-electrophoresis study also endorses that there is a strong affinity of the aqueous Shgel solution towards CT-DNA which is significant towards the formation of the stable DNA-hydrogel structure. Moreover, a fluorescence spectroscopy study shows that the luminescent DNA-hydrogel capped Ag-NPs are also fluorescent. The control experiments with pure CT-DNA in the photochemical synthesis of Ag-NPs justify the intriguing role of the DNA-hydrogel which acts as an efficient luminous capping agent to the fluorescent Ag-NPs.

Acknowledgements

B. D. is thankful to DST (New Delhi, India) for a research project (Project No.: SR/FT/CS-77/2011) for financial supports. S. P. S. B. is also thankful to CSIR, Govt. of India for a research grant (37 (1516/11/EMR-II)) for financial support.

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Footnote

† Electronic supplementary information (ESI) available: Schemes, ¹H and ¹³C NMR spectra of tetrapyridine, ESI-MS spectral patterns of tetrapyridine and Shgel, dynamic light scattering study, rheological patterns, hematological and serological parameters for toxicity evaluation, SEM image of the DNA-hydrogel, in vitro analysis, CD spectral study of the interactions of Ag-NPs with CT-DNA, fluorescence spectra of the Shgel, gel-documentation with UV-transillumination of the Shgel, fluorescence microscopy image of the Shgel, temperature dependent UV-absorption study of the Shgel, UV-absorption spectra of the Shgel, fluorescence micrograph of the Shgel and DNA-hydrogel, and fluorescence spectra and fluorescence micrograph of the DNA-hydrogel capped Ag-NPs. See DOI: 10.1039/c5ra19172f

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