Endogenous Activation-Induced Delivery of a Bioactive

Oct 19, 2016 - To gain some quantitative knowledge about the binding affinity of PSF with different hosts (micelle and ctDNA), we have exploited the f...
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Endogenous Activation-Induced Delivery of a Bioactive Photosensitizer from a Micellar Carrier to Natural DNA Mohd Afzal, Saptarshi Ghosh, Sinjan Das, and Nitin Chattopadhyay* Department of Chemistry, Jadavpur University, Kolkata 700032, India S Supporting Information *

ABSTRACT: The photophysics of phenosafranin (PSF), a member of the photosensitizer phenazinium family, has been explored in nonionic Triton X-165 (TX-165) micelle, calf thymus DNA (ctDNA), and a composite environment consisting of both micelle and DNA, using diverse spectroscopic techniques of both steady-state and timeresolved natures, to divulge the binding interactions of the probe with different hosts. The vivid experimental results demonstrate that PSF binds with both micelle and DNA; however, the binding affinity of the probe is much higher toward the DNA. When micelle-carried PSF comes in proximity to ctDNA, PSF gets released from the micellar environment and intercalates with the DNA base pairs. Endogenous activation, in terms of a higher binding affinity of PSF toward ctDNA relative to that toward the micelle, is ascribed to be responsible for this transfer. Thus, this article demonstrates endogenous transfer of a bioactive molecular probe from a micellar nanocarrier to natural DNA. As the carrier micelle (TX-165) does not perturb the structure of the DNA, this work proposes that it can be used promisingly for the purpose of safe delivery of drugs. The study is expected to stimulate the generation of and/or search for advanced micelle-based carrier systems for delivery of bioactive molecular systems to biological targets like DNA.

1. INTRODUCTION Paying emphasis on drug-induced adverse side effects in the present scenario, the most challenging task for therapeutic researchers is to discover efficient drug carriers that release drug molecules efficiently to the target of interest. Although a wide variety of nanosized materials have already been utilized for this purpose, the increasing need for such vehicles urges demanding research in the domain.1−5 Drug-delivery systems (DDS) have two advantages: they increase the efficacy of a drug through improved pharmacokinetics by controlling the biodistribution of the drugs and they reduce the drug-induced toxicity because the drugs are only active in the target zone (e.g., in cancerous tissues).1 Release of drug molecules from the DDS is generally controlled by two major approaches, namely, endogenous and exogenous activations.6 Endogenous activation utilizes some specific physicochemical characteristics of the drug as well as the microenvironment to release the carrier-loaded drug to the relevant target, whereas exogenous activation requires an external stimulus to initiate the drug-release process.6 Of course, it is of prime concern that neither the carrier nor the stimulant affects the structure of the target, to retain its normal functioning. Several reports are available in the literature on stimuli-responsive delivery of drugs/small molecules to the desired target, in which a variety of external stimuli, such as temperature, pH, light irradiation, magnetic ablation, ultrasound, and so forth, serve the purpose of inducing drug release.7−12 Unfortunately, in comparison with exogenous activation, endogenous release of drugs from the carrier to © XXXX American Chemical Society

the target is rather limited, despite the fact that it is devoid of the additional problems associated with an external stimulant.13,14 Micelles, formed by the aggregation of surfactant monomers, are one of the most commonly used drug carriers. Micelles can enhance the solubility and hence the bioavailability of drug molecules by accommodating them within their hydrophobic cores.15−18 Furthermore, the permeability and protective nature of micelles make them efficient drug carriers by assisting drug molecules to overcome the cell membrane barrier.19,20 On the receiving end, DNA is perhaps the most relevant biomolecular target, as it carries genetic information through its base sequence and is also involved in many important gene-related biological processes, like transcription and so forth.21−24 Drugs or small molecules are known to bind with DNA through three possible modes: (1) intercalation, where the molecule fits within the nucleic acid base pairs; (2) groove binding, where the molecule binds in either the deep major or shallow minor grooves of DNA via hydrogen bonding and/or van der Waals interactions; and (3) electrostatic binding, originating from mutual attractive interaction between the phosphate backbone of DNA and the cationic guest molecule.25,26 Among these three binding modes, intercalative binding is the most effective for drugs targeted to DNA.27 It is generally observed that most Received: August 16, 2016 Revised: September 20, 2016 Published: October 19, 2016 A

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been adopted for this purpose. Differential spectral responses of the probe in different microheterogeneous environments have been monitored to assess the location of the probe within the complex media. Experimental outcomes reveal that PSF binds strongly with both TX-165 micelle and ctDNA individually. However, upon addition of ctDNA to a solution of micellebound PSF, the probe is found to be dislodged from the micellar environment and intercalate with the DNA, resulting in relocation of PSF from the TX-165 micelle to the ctDNA. A higher binding affinity of PSF toward DNA compared to that toward the micelle is advocated as being responsible for the transfer of the probe from the micellar host to the DNA target. At the outset, CD study ensures that the carrier (TX-165 micelle) does not affect the structure of the target, projecting its safe use for the purpose. We believe that this study will help in developing other micelle-based delivery systems for efficient transfer of drugs/small molecules to DNA endogenously.

of the planar aromatic molecules favor intercalative binding, whereas the groove-binding mode is preferred for crescentshaped molecules.26,28 Alongside exploring the binding interactions between small molecules and DNA, carriermediated delivery of drugs to DNA has become a prospective topic of current biophysical research. In the present article, we have reported direct release of a small molecule, phenosafranin (PSF), from a micellar carrier to natural DNA through endogenous activation. PSF (Scheme 1), a member of the phenazinium family, has significant utility in various photophysical and photobiological applications, including photochemical sensitization.29 Nowadays, antimicrobial inactivation utilizing such sensitizers has become a promising method of photodynamic therapy (PDT).30 In this respect, cationic photosensitizers show significant efficiency against drug-resistant bacteria.31 Recently, Corio et al. have shown that PSF can be applied in PDT following functionalization with single-walled carbon nanotubes.32 Besides, phenazinium dyes are known to have antimalarial activity.33 Our group has shown that this potent biomolecular system (PSF) binds with calf thymus DNA (ctDNA) principally through the intercalative mode, although a minimal contribution of the electrostatic component was established from the effect of ionic strength of the solution on binding of PSF to DNA.26 Intercalation of PSF within DNA base pairs has also been confirmed by Das and Kumar through their viscosity study, thermal melting experiment, and circular dichroism (CD) spectral studies of both intrinsic and induced nature.34 Recently, quantitative release of PSF by exogenous means from an anionic micellar carrier to DNA has been demonstrated by our group.35 To avoid the complexity arising because of the involvement of multiple equilibria in the complex environment (carrier, target, and stimulant), in the present work, we have opted for a rather simple strategy using a nonionic Triton X-165 (TX-165) micellar carrier for transferring the probe endogenously to the most relevant biological target, DNA. Surfactants of the Triton X family contain a p-tert-octylphenyl hydrophobic moiety and a hydrophilic polyethyleneoxide chain (Scheme 1).36 Recently, Mazzoli et al. have exploited the Triton X-100 micelle for delivery of styryl drugs to DNA.37 Literature reports imply that nonionic surfactants are less toxic than ionic ones. 38 Furthermore, among structurally analogous surfactants, toxicity is known to decrease with an increase in the hydrophilic chain length or decrease in the hydrophobic chain length of the surfactant.38 Thus, among the members of the Triton X family, TX-165, possessing the longest hydrophilic chain, is reported to be the least toxic one.39 To demonstrate the delivery of PSF from micelle to DNA, we have investigated the interactions of PSF with the micelle and ctDNA separately. Thereafter, the effect of ctDNA on the probe−micelle complex has been explored. Vivid steady-state and time-resolved spectroscopic techniques along with CD and DNA helix melting studies have

2. EXPERIMENTAL SECTION 2.1. Materials. PSF, TX-165, and bovine serum albumin (BSA) (all from Sigma-Aldrich); Tris−HCl buffer and ctDNA (both from SRL, India); and spectroscopic-grade 1,4-dioxane (Spectrochem, India) were used without further purification. The ctDNA sample has a molecular weight of 8.4 MDa. Deionized water obtained from a Milli-Q (Millipore) water purifier was used throughout. During all of the measurements, the medium used was 0.01 M Tris−HCl buffer, with a pH of 7.4. ctDNA solutions were prepared following the procedure described in the recent literature.35,40,41 Unless otherwise specified, the PSF concentration was maintained at ∼5 μM for the entire study. Freshly prepared micellar solutions were used to avoid aging-related problems. 2.2. Methods. Absorption measurements were carried out on a Shimadzu UV-2450 spectrophotometer (Shimadzu Corporation, Japan), whereas steady-state fluorescence and fluorescence anisotropy measurements were performed with a Horiba Jobin Yvon Fluoromax-4 spectrofluorometer. A FluoroCube instrument (Horiba Jobin Yvon), with a laser diode (IBH, UK) with an output of 490 nm and a TBX photon detector, was used for the time-resolved fluorescence decay measurements, adopting the time-correlated single photon counting technique.42 The technical details regarding steadystate fluorescence anisotropy and fluorescence lifetime measurements have already been described in some of our earlier publications.35,41 A JASCO J-815 spectropolarimeter (Jasco International Co., Japan) was used for recording the CD spectra, using a regular rectangular quartz cuvette with a 1 cm path length and with a scan speed of 100 nm min−1. Proper baseline corrections were performed against the buffer solution to extract the corrected CD spectra. B

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The Journal of Physical Chemistry B For helix melting experiments, a high-precision Peltier (Wavelength Electronics), attached to the absorption spectrophotometer, was used to set the prefixed temperature. Sufficient time was allowed before each measurement to affirm attainment of thermal equilibrium throughout the solution. Dynamic light scattering (DLS) measurements were performed using a Nano-ZS90 Malvern instrument (Model DLS-nano ZS, Zetasizer, Nano series) equipped with a 4 mW He−Ne laser (λ = 632.8 nm) and a thermostatic sample chamber. The hydrodynamic sizes of the samples were measured using associated instrument software. The samples were filtered through a 0.22 μm syringe filter before the measurements to remove the dust particles.

3. RESULTS AND DISCUSSION 3.1. Verification of Retention of the DNA Structure in the Presence of TX-165 Micelle. To ensure that the TX-165 carrier micelle does not affect the structure of the ctDNA, we have monitored the structural aspects of the DNA through its intrinsic CD spectra in the absence and presence of different TX-165 concentrations. Figure 1 represents the far-UV CD

Figure 2. Variation in the absorption spectra of PSF with increasing concentrations of TX-165. Curves (i) → (xi) correspond to [TX-165] = 0, 0.39, 0.69, 1.95, 2.67, 4.52, 6.96, 10.29, 13.01, 15.58, and 16.28 mM. [PSF] = 5 μM.

significant modification in the absorption spectra of the probe in the micellar medium. Gradual addition of TX-165 to the aqueous buffered solution of PSF results in a decrease in absorbance together with a bathochromic shift in the band maximum by 10 nm (from 520 nm in the aqueous buffer to 530 nm in the TX-165 micellar medium). The observed red-shift is the signature of lowering of the polarity of the immediate environment around PSF in the micellar medium compared to that in the aqueous buffer milieu. The bathochromic shift in the absorption maximum of the probe is consistent with our previous observation in other micellar media.35 In a similar manner, successive addition of ctDNA to the buffered solution of PSF leads to a decrease in the absorbance, associated with a larger shift in the absorption maximum, from 520 to 540 nm (Figure S1 in the Supporting Information). The absence of a distinct isosbestic point in the absorption spectra of PSF in the presence of varying concentrations of the DNA rules out intercalation of PSF within ctDNA to be the sole binding interaction.26,35 Successive addition of ctDNA to micelle-bound PSF results in a decrease in the absorbance, accompanied by a hyperchromic shift of the absorption maximum by about 10 nm (from 530 nm in the micellar medium to 540 nm upon addition of 0.41 mM ctDNA). The variation in the absorption spectra of micelle-bound PSF with increasing concentration of ctDNA is presented in Figure 3. The spectral modifications in Figure 3 suggest that in the composite medium (TX-165 micelle + ctDNA) PSF experiences an environment different from the one in the presence of TX-165 micelle alone. In the presence of a high enough ctDNA concentration, the position of the absorption maximum of the probe coincides with that observed in a medium with only DNA (540 nm). For a clearer visualization of the positions of the absorption maxima of PSF in different media, we have presented some normalized spectra in Figure 4. Agreement of the position of the absorption maximum of the micelle-bound probe in the ctDNA medium with that of the probe in the medium with only DNA suggests that PSF experiences similar environments in these two situations. Thus, the spectrophotometric studies reveal that in the composite medium (micelle + ctDNA) PSF prefers to reside selectively within the DNA environment. The discussion in the following sections will substantiate this explicitly. 3.3. Steady-State Fluorometric Studies. To study the photophysical behavior of PSF in TX-165 micelle, ctDNA, and

Figure 1. Intrinsic CD spectra of ctDNA in buffer (green) and in the presence of different concentrations of TX-165, as mentioned in the legends. [ctDNA] = 100 μM.

spectrum of ctDNA in buffer solution in the absence and presence of two different concentrations of TX-165, one below the critical micelle concentration (CMC) and one above the CMC (the CMC of TX-165 is 0.43 mM in aqueous medium36). The CD spectrum of DNA in an aqueous buffer medium shows a characteristic positive peak at ∼274 nm and a negative peak at ∼244 nm, signatures of the B form of DNA.26 Figure 1 demonstrates that the CD spectrum remains indifferent to the addition of surfactant or TX-165 micelle, emphasizing that by no means do they affect the secondary structure of ctDNA. Thus, retention of the structure of the DNA in the presence of TX-165 micelle convincingly proposes that this micelle can be employed as safe carrier for drugs or small molecules targeted to DNA. 3.2. Spectrophotometric Studies. To have an idea about the interaction of different microheterogeneous environments with the probe in the ground state, absorption spectra of PSF have been recorded in TX-165 micellar and ctDNA environments. In an aqueous buffer medium, PSF shows a low-energy unstructured absorption band peaking at 520 nm. The absorption spectra of PSF at different TX-165 concentrations are presented in Figure 2. As evident from Figure 2, there is C

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the micelle−water interfacial region rather than to enter the nonpolar micellar core. We have carried out a DLS study to see whether there is a drastic change in the morphology of the micelle upon binding with PSF. The study revealed that binding with the probe leads to inappreciable modification in the size of the TX-165 micelle (Figure S2). Variation in the fluorescence spectra of PSF in the presence of added TX-165 is depicted in Figure 5A. On the contrary, upon binding with the DNA, the intensity of emission of PSF decreases remarkably, with a less significant (∼7 nm) blue-shift of the emission maximum (Figure S3). The fluorometric responses of PSF in both the media agree well with the literature reports in other micellar media and ctDNA environment, depicting strong binding interactions of PSF with both the TX-165 micelle and DNA.26,44 The variation in the steady-state emission intensity and fluorescence lifetimes (see later) in the micellar as well as ctDNA environment relative to those in an aqueous buffer medium can be rationalized from the radiative (kr) and nonradiative (knr) rate constants, as determined from the fluorescence quantum yields (Φf) and radiative lifetimes (τ) of the fluorophore in various media (Table S1). The reduction in the fluorescence intensity of the probe in a DNA environment is due to the decrease in the rate of the radiative decay process of the probe in a DNA medium compared to that in the aqueous buffer milieu. To gain some quantitative knowledge about the binding affinity of PSF with different hosts (micelle and ctDNA), we have exploited the fluorescence titration data and modified Benesi−Hildebrand equation45 to determine the binding constants with TX-165 micelle and ctDNA. The relevant equation is

Figure 3. Absorption spectra of micelle-bound PSF with addition of ctDNA. Curves (i) → (xii) correspond to [ctDNA] = 0, 0.002, 0.007, 0.012, 0.024, 0.036, 0.048, 0.065, 0.084, 0.15, 0.32, and 0.41 mM. [PSF] = 5 μM.

1 1 1 1 = + ΔF ΔFmax K ΔFmax [L] Figure 4. Absorption spectra of PSF under different solution conditions, as mentioned in the legends.

where ΔF = Fx − F0 and ΔFmax = F∞ − F0, with F0, Fx, and F∞ being the fluorescence intensities of PSF in the absence of micelle or DNA, at an intermediate micellar or DNA concentration, and at a concentration for complete interaction, respectively; K is the binding constant and [L] the micellar or DNA concentration. According to Almgren et al.,46 the micellar concentration is calculated as

the composite medium and to thereby understand the binding interactions of PSF in different situations, steady-state fluorometric studies have been performed. In buffer, PSF gives a single broad charge transfer emission band peaking at 582 nm.43 With the addition of TX-165 to the aqueous buffered solution of PSF, the fluorescence intensity of the probe increases drastically, with a significant hypsochromic shift of 16 nm (from 582 nm in the buffer to 566 nm in ∼16 mM TX-165 micelle), depicting strong binding interaction of the probe with the micelle. Being ionic in nature, PSF should prefer to bind at

[L] = (S − CMC)/N

where S represents the surfactant concentration and N is the aggregation number of the micellar system. The aggregation number of TX-165 micelle in Tris buffer has been determined following the standard fluorescence quenching method,47 and it

Figure 5. (A) Variation in the emission spectra of PSF with the addition of TX-165. [PSF] = 5 μM, λex = 520 nm. (B) Double-reciprocal plot for the complexation between PSF and TX-165. D

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Figure 6. (A) Fluorescence spectra of micelle-bound PSF with the addition of ctDNA. [PSF] = 5 μM, λex = 520 nm. (B) Fluorescence spectra of PSF in different media, as mentioned in the legends.

establish the stability of PSF-carrying micelle in the presence of BSA. Distinct fluorometric behaviors of PSF in TX-165 micelle and DNA environments have been explored to assess its precise location in the composite medium. With gradual addition of ctDNA to the micelle-bound probe, the emission intensity of the probe decreases drastically, with a hyperchromic shift of the band maximum. The variation in the fluorescence spectra of micelle-bound PSF with gradual addition of ctDNA is presented in Figure 6A. At a high enough DNA concentration, the emission intensity matches the intensity of the probe in the medium with only ctDNA. For a better perception, we have presented the emission spectra of PSF in different environments in Figure 6B. The emission maximum of PSF in the composite medium (micelle + ctDNA) practically coincides with that of the probe in the DNA medium. Therefore, the fluorometric responses of micelle-bound PSF on addition of ctDNA indicate that in the presence of DNA the probe gets released from the TX-165 micelle and binds to the DNA. The relocation of the probe from the micelle to the DNA is rationalized by the consideration of a higher binding affinity of the probe toward DNA compared to that with the micelle, leading to a simple case of competitive binding. We have confirmed the location of the probe in the medium containing both micelle and DNA from several other studies described in the forthcoming sections. 3.4. Micropolarity Study. An effective approach to assess the specific location of the fluorophore in a complex microheterogeneous environment is to determine the polarity of the immediate vicinity of the probe.28,41,48 The micropolarities around the fluorophore in microheterogeneous environments are determined with the help of the ET(30) scale, as developed by Kosower and Reichardt.49,50 The fluorometric responses of the fluorophore in the concerned environments are compared to those in a series of homogeneous solvent mixtures of known ET(30) values. To judge the location of PSF in the composite medium (micelle + ctDNA) of the present work, we have determined the micropolarities of PSF in TX-165 micelle, ctDNA, and the composite medium separately. At the outset, a calibration plot monitoring the energies corresponding to the emission maxima of PSF in water−dioxane mixtures of varying compositions with known polarities was constructed (Figure 7). The ET(30) values for different compositions of dioxane−water mixtures are obtained from the work of Kosower et al.49 It is pertinent to mention here that the water−dioxane mixture is chosen for constructing the calibration plot over other common

was found to be 75. The corresponding plot is provided in Figure S4. From the Benesi−Hildebrand double-reciprocal plot (Figure 5B), using the fluorescence titration data of PSF in the TX-165 micellar medium, the binding constant of the probe with the micelle is determined to be 1.1 × 104 M−1. From the determined binding constant (K) value, the free-energy change for this PSF−TX-165 binding is estimated to be ΔG = −23.05 kJ mol−1, indicating the process to be thermodynamically feasible and spontaneous. In a similar manner, the binding constant of PSF with ctDNA is determined to be 5.6 × 104 M−1 (Figure S5). The high binding constant for the binding of the probe with DNA can be justified by considering the intercalative binding of PSF with ctDNA; as for intercalation, the binding constants are generally estimated to be in the order of ∼104−105 M−1.26,34 The determined binding constants reveal that PSF has strong binding interactions with both the TX-165 micelle and ctDNA independently, notwithstanding the binding constant being remarkably higher with the DNA than with the micelle. Hence, it is expected that in the presence of both ctDNA and TX-165 micelle PSF should prefer to bind to the former. In one of our earlier studies, we successfully demonstrated the transfer of PSF from anionic sodium tetradecyl sulfate (STS) to DNA.35 As the binding affinity of PSF toward STS (∼106 M−1) is orders of magnitude higher than that toward ctDNA, we intelligently used β-cyclodextrin as an external stimulant to induce the release of PSF by disrupting the micellar structure. The relative magnitudes of the binding constants of PSF with TX-165 and ctDNA prompted us to go for endogenous transfer of the probe from the micelle to the DNA, avoiding an external stimulant. The least toxicity of the TX-165 micelle compared with other nonionic micellar systems of the Triton X family also contributes toward its selection as the carrier. Before exploring the behavior of the PSF−micelle complex in the presence of ctDNA, it is important to look at the stability of the former in the presence of BSA, the most abundant serum protein in the circulatory system. For this purpose, the steadystate as well as time-resolved fluorometric response of the micelle-bound probe has been investigated with the addition of BSA (Figures S6). As evident from Figure S6A, addition of BSA to the TX-165 micelle-bound probe does not lead to a significant change in the fluorescence intensity (the marginal decrease arises from the dilution effect). Similarly, the timeresolved fluorescence decay pattern of the micelle-bound dye is also not affected significantly upon addition of BSA (Figure S6B). These observations clearly imply that BSA does not affect the probe−micelle binding meaningfully. These experiments E

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3.5. Fluorescence Anisotropy Measurements. Steadystate fluorescence anisotropy measurements report on the motional restriction experienced by the fluorophore from its neighboring environment; thus, it is utilized to judge the location of the fluorophore in microheterogeneous environments like micelles, reverse micelles, lipids, proteins, cyclodextrins, DNA, and so forth.28,36,42−44 Factors affecting the size, shape, and segmental flexibility of a molecule affect the observed fluorescence anisotropy.42 An increase in the rigidity of the environment surrounding the probe results in an increase in the fluorescence anisotropy.42−44 In this case, we have measured the anisotropy value of the probe in different media to judge its location in these media. Variations in the fluorescence anisotropy values of PSF in different environments are depicted in Figure 8, and the significant data are compiled in Table 2. Table 2 reveals that the fluorescence anisotropy of

Figure 7. Calibration plot for the determination of ET(30) values of PSF in different solutions. The open circles give the interpolated energies corresponding to the emission maximum values in different environments, as mentioned in the legends.

Table 2. Fluorescence Anisotropy of PSF in Different Environments

homogeneous solvent mixtures, like water−alcohol, because of the fact that the water−dioxane mixture covers a much wider range of polarity.28,48 The ET(30) values around PSF in different media are determined by interpolating the energies corresponding to the emission maxima of the probe in these media on the calibration line (Figure 7). The ET(30) values extracted from the plot are collected in Table 1. The values

ET(30) (kcal mol−1) (±0.2)

buffer TX-165 (16.28 mM) DNA (0.41 mM) TX-165 (16.28 mM) + DNA (0.41 mM)

60.9 50.8 56.2 54.9

anisotropy (r) (±0.005) 0.034 0.179 0.295 0.276

PSF in TX-165 micelle and ctDNA environments are significantly higher than that in an aqueous buffer medium, indicating imposition of some sort of rotational restriction on PSF upon binding with both the hosts. A much higher anisotropy value of the probe in ctDNA is attributed to the intercalative binding of the probe with ctDNA.26 With gradual addition of ctDNA to micelle-bound PSF (latter part of Figure 8), the anisotropy of the probe increases steadily, and at a high enough DNA concentration, the value approaches 0.28, close to the value obtained when the probe intercalates within the DNA base pairs (Table 2). This observation demonstrates that the probe experiences similar motional restrictions from its surrounding microenvironment in DNA as well as in the composite medium (micelle + DNA). Thus, fluorescence anisotropy measurements provide additional support in favor of the relocation of the probe from the TX-165 micelle to ctDNA.

Table 1. Micropolarity Values of PSF in Different Media environments

environments buffer TX-165 (16.28 mM) DNA (0.41 mM) TX-165 (16.28 mM) + DNA (0.41 mM)

reveal that the micropolarities around PSF in all three microheterogeneous media are significantly less compared to those in the aqueous buffer medium, implying that the probe resides in rather hydrophobic regions in these assemblies. Inspection of the micropolarity values further discloses that PSF shows similar polarities in the composite medium and the ctDNA environment, reiterating that relocation of the micellebound PSF to the DNA takes place in the presence of both the hosts.

Figure 8. Variation in the fluorescence anisotropy of PSF with increasing concentrations of TX-165 and in that of the micelle-bound probe with increasing DNA concentrations. The inset shows the variation of anisotropy in DNA environments. λex = 520 nm and λmonitor = λmax em in the respective medium. Each data point is an average of 15 consistent individual measurements. F

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fluorescence decay of the probe in the composite medium agrees well with that in the medium with only DNA. This agreement implies similar microscopic environments around the probe in the two situations and thereby supports the transfer of the probe from the TX-165 micelle to ctDNA. 3.7. CD Study. To analyze the nature of the microenvironment and hence substantiate the transfer of PSF from the micellar medium to the DNA, we have performed CD spectral measurements as well. PSF, being an achiral and planar molecule, does not show any CD spectrum of its own.26 Upon binding with ctDNA, however, PSF shows a strong positive peak at around 550 nm, along with a weaker negative peak at around 510 nm. It is to be remembered that ctDNA has no CD signal in the wavelength region of 400−600 nm. Hence, the development of the two CD bands in the visible region is rightfully ascribed to the imposed asymmetry on the probe because of its intercalation within the DNA base pairs.26,34 Similar kinds of induced CD bands have also been observed for ethidium bromide, a well-known DNA intercalator, upon binding with ctDNA.52 Induced CD spectra for DNA binders originate because of the coupling of the electric transition dipoles of the binder molecule and DNA bases within the asymmetric DNA environment.52 The very low concentration of the probe in the solution (in the micromolar range) rules out the possibility of aggregate formation by the probe on the DNA surface, as PSF forms aggregates only at a much higher concentration (in the millimolar range).53 The induced CD spectra of PSF with increasing DNA concentrations are presented in Figure 10A. In the TX-165 micellar medium, we do not obtain any induced CD spectra of the probe (Figure 10B), as expected. Interestingly, with gradual addition of DNA to the micelle-bound PSF, CD signals, as already described, develop in a similar manner to that in the ctDNA environment (Figure 10B). Therefore, the present observations imply that in the presence of DNA the micelle-bound PSF relocates from the micellar medium to the DNA environment. 3.8. DNA Melting Experiment. To confirm the intercalation of PSF in ctDNA as well as in the composite medium and to thereby ensure the transfer of the probe from the micellar carrier to ctDNA, we have further adopted the DNA melting experiment. During the DNA melting process, the nonbonding interactions of the double helix are ruptured by increasing the temperature of the solution, resulting in disruption of the helical structure of the DNA.54 The melting temperature (Tm) of DNA is defined as the temperature at which half of the double-helical DNA strands are unfolded to single strands.55 The molar extinction coefficient (at 260 nm) of double-helical DNA is significantly less than that of its singlestranded form.28,48 The helix melting temperatures of DNA in different situations are determined from the inflection points of the sigmoidal curves obtained by measuring the absorbance at 260 nm as a function of temperature. The melting profiles of ctDNA in different media are presented in Figure 11, and the melting temperatures are compiled in Table 4. It is pertinent to mention here that in the present experiment we have used a relatively lower concentration of ctDNA and a higher concentration of PSF compared to those used in the other studies performed, to ensure that all of the DNA molecules are engaged in intercalation with the PSF molecules. The melting temperature (Tm) of DNA, however, does not depend on the concentration of DNA. The melting temperature of native DNA in aqueous buffer is estimated to be 71.0 ± 0.3 °C, consistent with the literature

3.6. Time-Resolved Fluorescence Study. Fluorescence lifetime is another important parameter that is sensitive to the surrounding medium and also to the excited-state interactions of the probe, which provides important information about the residence of the probe within microheterogeneous assemblies.35,36,40,42,48 In the present case, we have analyzed the timeresolved fluorescence decays of the probe in different media to establish its relocation from micelle to the DNA in the composite medium. The fluorescence decay profiles of PSF in TX-165 micellar media and ctDNA environments are provided in Figures S7 and S8, respectively, and the corresponding deconvoluted data are presented in Tables S2 and S3, respectively. In both aqueous buffer and micellar media, PSF yields single exponential decays, indicating the residence of the probe in a single environment.44 With increasing concentrations of both TX-165 and ctDNA, the fluorescence lifetime of PSF increases significantly, confirming the binding of the probe to both the assemblies.51 Although the lifetimes of the probe in both the media are of similar orders, there is a fundamental difference in the fluorescence decays in the two environments, being biexponential in the ctDNA medium and single exponential in the TX-165 medium. Multiexponential decays of fluorophores are frequently observed in biological or biomimicking assemblies and are ascribed to originate from multiple locations of the environment differing in polarity.28,35,36,42 To avoid the complexities associated with putting much emphasis on the individual decay components, it is rather convenient to use average fluorescence lifetimes to understand the behavior of fluorophores within microheterogeneous assemblies, and this is often adopted.28,35,36,48 On gradual addition of ctDNA to micelle-bound PSF, the decays become visibly biexponential and at a high enough DNA concentration, the decay profile of PSF becomes almost the same as that in an environment with only ctDNA. The decay profiles of micellebound PSF on addition of DNA are presented in Figure S9, and the corresponding decay parameters are tabulated in Table S4. For easy inspection of the changes in the decay patterns of PSF with a change in the environment, we have plotted the decay profiles of PSF selectively in buffer, micelle, DNA, and the composite medium in Figure 9, and the lifetime parameters in these media are compiled in Table 3. A glance at Table 3 reflects that the complete set of analyzed data for the

Figure 9. Time-resolved fluorescence intensity decays of PSF in different environments, as labeled in the legends. The sharp profile (black) on the left is the lamp profile (instrument response function). [PSF] = 5 μM, λex = 490 nm, and λem = λmax em . G

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a1

τ1 (ns)

buffer TX-165 (16.28 mM) ctDNA (0.41 mM) 16.28 mM TX-165 + 0.41 mM ctDNA

1 1 0.78 0.77

0.88 1.85 1.24 1.30

a2

0.22 0.23

τ2 (ns)

2.65 2.61

τavg (ns)

χ2

1.55 1.60

1.08 1.16 1.12 1.02

Figure 10. (A) Induced CD spectra of PSF in different ctDNA environments in the absence of TX-165 micelle. (B) Induced CD spectra of micellebound PSF in different ctDNA environments. The concentrations of ctDNA in the different environments are provided in the legends. [PSF] = 5 μM.

described in Section 3.1, that TX-165 micelle have an insignificant effect on the structure and stability of DNA (Figure 1). However, addition of ctDNA to micelle-bound PSF results in an appreciable increase in the Tm value (75.5 ± 0.3 °C). The agreement of the Tm value of DNA in the presence of PSF in the composite medium with that of DNA intercalated with PSF (Table 4) unambiguously reveals that the probe remains within the DNA base pairs in the composite medium and hence confirms the delivery of PSF from the TX-165 micelle to DNA.

4. CONCLUSIONS The present study unequivocally demonstrates the delivery of bioactive PSF molecules to natural DNA through a nonionic TX-165 micellar carrier. Vivid steady-state and time-resolved spectroscopic measurements along with helix melting study of DNA have been conducted to establish the relocation of PSF from the micellar environment to DNA. The estimated binding affinities of PSF with TX-165 micelle and ctDNA imply that the molecule binds strongly with both these systems, although the affinity toward DNA is 5 times higher than that toward the micelle. The experimental results confirm that with the addition of ctDNA to micelle-bound PSF, relocation of the probe takes place from the TX-165 micelle to the DNA. The higher binding affinity of PSF with ctDNA relative to that with the micelle accounts for this transfer. DNA melting and induced CD studies corroborate that in the composite medium consisting of both micelle and DNA, PSF remains intercalated within the DNA base pairs. At the outset, intrinsic CD study has been adopted to reveal the nonvicious nature of TX-165 micelle toward ctDNA, as far as structural aspects are concerned. This observation, therefore, projects that this micellar system can be safely exploited as a nanocarrier for drugs or small molecules targeted toward biological entities, like DNA.

Figure 11. DNA melting profiles under different conditions, as described in the legends. [ctDNA] = 50 μM, [PSF] = 20 μM, and [TX-165] = 1.72 mM.

Table 4. Melting Temperatures (Tm) of ctDNA in Different Media environments

Tm (±0.3 °C)

native DNA DNA + PSF DNA + TX-165 DNA + TX-165 + PSF

71.0 75.6 71.3 75.5

value.48 Upon addition of PSF to the DNA solution, the melting temperature increases significantly and is determined to be 75.6 ± 0.3 °C. The appreciable increase in the Tm value divulges intercalation of the probe in the DNA double helix, as it is already known that intercalation of molecules within the DNA base pairs increases the stability of the helix, resulting in an appreciable increment in the melting temperature, whereas other modes of binding cause imperceptible changes in the Tm.27,28,34,35,48 Addition of TX-165 to the buffer solution of DNA yields practically no change in the melting temperature (71.3 ± 0.3 °C), supporting the results of the CD study, as

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b08283. H

DOI: 10.1021/acs.jpcb.6b08283 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B

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Absorption of PSF in different ctDNA environments; DLS data of TX-165 in the absence and presence of PSF; emission spectra of PSF with increasing DNA concentrations; plot for determination of the aggregation number of TX-165 micelle; double-reciprocal plot of PSF with ctDNA for the determination of the binding constant with ctDNA; emission spectra and timeresolved fluorescence decay of micelle-bound PSF with increasing BSA concentrations; time-resolved fluorescence decay profiles of PSF in different environments; photophysical and deconvoluted time-resolved parameters for PSF in micelle, ctDNA, and the composite medium (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 919433948648. Fax: 91-33-2414-6584. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from C.S.I.R. (Project No. 01(2807)/14/ EMR-II), Govt. of India, is gratefully acknowledged. The authors thank Dr. G. Suresh Kumar and Pritha Basu of CSIRIICB, Kolkata, for lending the CD instrument and experimental assistance respectively. They also thank Prof. S.C. Bhattacharya of Jadavpur University for allowing the authors to perform the DLS study. M.A. thanks U.G.C. for the Dr. D.S. Kothari Postdoctoral Fellowship (BSR/CH/15-16/011). S.G. and S.D. thank the same funding agency for providing them with research fellowships.

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