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. 2019 Sep 17;4(14):15829–15841. doi: 10.1021/acsomega.9b01543

Probing the Intercalation of Noscapine from Sodium Dodecyl Sulfate Micelles to Calf Thymus Deoxyribose Nucleic Acid: A Mechanistic Approach

Neha Maurya †, Khalid Ahmed Alzahrani ‡, Rajan Patel †,*
PMCID: PMC6777008  PMID: 31592453

Abstract

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Noscapine (NOS) is efficient in inhibiting cellular proliferation and induces apoptosis in nonsmall cell, lung, breast, lymphatic, and prostate cancers. The micelle-assisted drug delivery is a well-known phenomenon; however, the proper mechanism is still unclear. Therefore, in the present study, we have shown a mechanistic approach for the delivery of NOS from sodium dodecyl sulfate (SDS) micelles to calf thymus deoxyribose nucleic acid (ctDNA) base-pairs using various spectroscopic techniques. The absorption and emission spectroscopy results revealed that NOS interacts with the SDS micelle and resides in its hydrophobic core. Further, the intercalation of NOS from SDS micelles to ctDNA was also shown by these techniques. The anisotropy and quenching results further confirmed the relocation of NOS from SDS micelles to ctDNA. The CD analysis suggested that SDS micelles do not perturb the structure of ctDNA, which supported that SDS micelles can be used as a safe delivery vehicle for NOS. This work may be helpful for the invention of advanced micelle-based vehicles for the delivery of an anticancer drug to their specific target site.

1. Introduction

Over the past few decades, drug–DNA interactions have gained interest in clinical applications and cancer research.1,2 A brief knowledge about the mechanism of drugs, such as identification a drug molecule and its reversible binding to DNA, is important for rational drug design and drug delivery. It is necessary to gain information on drug–DNA complexes, binding mechanism, dynamics of complex formation, and driving energies of the process. Mainly two types of noncovalent interactions involve in drug–DNA binding, groove binding and intercalation.3 Furthermore, the information on specific drug delivery and the efficient drug released are of great importance for pharmaceutical and drug development research. Presently, poor bioavailability of anticancer drugs is a serious challenge in pharmaceutical industry and cancer research. To resolve this issue, surfactant systems are used as attractive vehicles for drug delivery.4,5 Because of its hydrophobic nature and electric charge, the drug molecule can be solubilized into the core of the micelle or at the surface or intermediate location in the palisade layer. In-depth understanding about the mechanism of molecular interaction between drugs and surfactants and their release to the target site is a significant aspect in the formulation of a new drug molecule and its effective delivery.6 Solubilization of the drug in micelles results in the enhancement of bioavailability and water solubility and eliminates other adverse effects.7,8 Apart from the natural resemblance with the biomimic models, micelles are spectroscopically silent and scatter-free, accumulate in the required area, and remain optically transparent. Therefore, in modern research, fluorophore drugs in the presence of the micelles provide molecular level dynamics using spectroscopic techniques.8

Noscapine (NOS) is a phthalideisoquinoline alkaloid extracted from Papaver somniferum plant. It is antitussive and a naturally tubulin-binding compound presently undergoing phase I/II clinical trials for cancer treatment.9,10 Because of low bioavailability and poor physiochemical properties, the therapeutic concentration of NOS cannot be attained at the targeted sites. Additionally, NOS is eliminated from plasma, visceral organs, and circular system through first-order elimination rate constant, resulting in low accumulation of NOS in tumor cells.11,12 To enhance the bioavailability, recently, Madan and co-workers improved sterically stabilized gelatin micro assemblies of NOS12 and also reported inhalable nanostructured lipid particles of 9-bromo-NOS for drug delivery of NOS.13 Sebak et al.14 worked on human serum albumin (HSA) nanoparticles as an efficient NOS drug delivery system for the potential use in breast cancer. More recently, we have shown the effect of NOS on the activity and structure of HSA.15 In addition to this, herein, we have tried to find out some new targets for NOS, and therefore, we have studied the NOS–calf thymus deoxyribose nucleic acid (ctDNA) interaction. Moreover, to improve the bioavailability of NOS, we have also shown the encapsulation of NOS in sodium dodecyl sulfate (SDS) micelles and their intercalation with ctDNA from SDS micelles. In this study, we have taken SDS (an anionic surfactant) as a model surfactant which also behaves as a model membrane structure, where NOS associates into the core of the micelle of the surfactant. In the past decades, Mitra et al.16 reported the nature of binding of nile blue with SDS micelles, AOT reverse micelles, and genomic DNA from salmon sperm. Recently, Mazzoli et al.17 showed the interaction of new potential anticancer drugs with DNA in the nonionic micellar system. In this perspective, in the present study, we have worked on the interaction of NOS with ctDNA in the presence of anionic micellar assemblies (SDS) and we have also investigated the interaction of NOS with SDS micelles using spectroscopy techniques. Scheme 1 shows the systematic representation of the intercalation of NOS from SDS micelles to ctDNA.

Scheme 1. Systematic Representation of the Intercalation Process.

Scheme 1

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2. Result and Discussion

2.1. Absorption and Emission Spectral Studies

2.1.1. Interaction of NOS with SDS Micelles

The structure of NOS is composed of two aromatic cyclic rings: a phthalide ring with S configuration and an isoquinoline ring with R configuration. These two types of rings are linked through chiral carbons (Scheme 2).22 They have two stereogenic centers and therefore consist of four possible stereoisomer carbons.23Scheme 2 shows the structure of NOS.

Scheme 2. Structure of NOS.

Scheme 2

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The absorption and emission spectra of NOS were observed because of π–π* transition between these two rings. In the absorption measurement of NOS, two absorption bands were observed at 290 and 312 nm in the aqueous buffer solution (Figure S1A). These two absorption bands were practically dependent on the nature of solvent polarity. NOS showed a single emission peak in nonpolar solvents and a dual emission peak in the polar solvent. The emission peaks were found at 402 and 467 nm in aqueous buffer solution (Figure S1B) and the single peak was found at 373 nm in dimethyl sulfoxide. It is known from the literature that NOS is less soluble in water and thus SDS micelles are used to enhance its solubility and therefore help its delivery to the target site.19 To facilitate the spectral properties of NOS in the micellar microenvironment, the absorption and emission spectra of the molecule were investigated in the anionic micelle environment. Figure 1a shows the absorption spectral properties of NOS in the aqueous buffer medium at physiological pH (7.0) with an increasing quantity of anionic surfactant SDS from precritical micelle concentration (cmc) to post cmc concentration (0.2–10 mM). Upon increasing the SDS concentration, the intensity of both absorbance bands was gradually enhanced with a slight blue shift (290–288 and 312–308 nm) and with a shoulder at 320 nm at the micellar and above micellar concentration range (Figure 1A).The hyperchromic and hypsochromic shifts in the presence of SDS micelles clearly suggest increased solubility of NOS in the micellar environment due to the nonpolar environment of SDS micelles.24 The presence of a shoulder at a higher concentration of SDS suggested that NOS is present in a vastly different environment and a strong interaction between SDS micelles and NOS occur in the stern layer. Figure 2 shows the relative change in the absorbance of NOS at 312 nm against the pre to post micellar concentrations of SDS. Initially, a slight increase in the absorbance of NOS shows the involvement of the electrostatic interaction, while the large hyperchromic shift in the absorbance around cmc suggested that NOS is entrapped into the hydrophobic core of the micelle which may be due to the hydrophobic–hydrophobic interaction and at high concentrations, that is, above micellar concentration, the absorbance remains constant.5 From Figure 2, it can be seen clearly that the cmc of SDS in the presence of NOS was decreased and found at around 2.4 mM in the aqueous buffer medium at room temperature. The cmc of SDS (8.2 mM) may be varied with different external factors such as pH, salt concentration, temperature, buffer condition, and interaction with drugs. Makowska et al. reported that different salt concentrations also change the cmc of SDS.25 Another literature also showed a decrease in the SDS cmc in the presence of the drug.5 This information suggests that NOS is located inside the hydrodynamic sphere of micelles and goes to a more nonpolar environment than its previous state.

Figure 1.

Figure 1

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(A) Absorption spectra of NOS (50 μM) in different concentrations of SDS (0.2–10 mM). (B) Absorption spectra of NOS under different system.

Figure 2.

Figure 2

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Plots of variation of relative absorbance and relative fluorescence intensity of NOS in the presence of SDS.

Emission spectral analyses were also carried out to understand the behavior of NOS in SDS micelles. The emission spectrum of NOS shows very weak intensity around 467 nm with a shoulder at 402 nm, when exited at 312 nm. We obtained a very interesting result after adding SDS. Emission spectra of NOS at 467 nm were decreased with the addition of a lower concentration of SDS (up to 0.8 mM), but at higher concentrations, the intensity increases gradually. The shoulder peak at 402 nm turns to a complete specific peak and appears at 394 nm with a blue shift of 8 nm and the peak at 467 nm disappeared around cmc, that is, 2.4 mM (Figure 3A and 3B). The quenching of NOS emission by a lower SDS concentration may be caused due to the formation of NOS and monomer surfactant ion-pair interactions which increasingly associate to form an aggregated type of structure as micelles.26 At a higher concentration of SDS, the fluorescence intensity of NOS increases. This is because as the fluorophore of NOS interacts with SDS at higher concentrations, the structure of NOS is altered; moreover, the environment around the fluorophore molecule changes from high-polarity to a low-polarity region. Also, the rotational movement of the free NOS molecules favors a radiation-less deactivation of the excited states in aqueous solution.27 However, after binding with SDS molecules, the rotation is hindered and the deactivation occurs through fluorescence emission, which may be responsible for the significant increase in the fluorescence intensity of NOS. A large blue shift also specifies the location of the fluorophore in the low polar region of micelles, with the hydrophobic moiety of the probe intruded into the hydrocarbon core region of the micelle. A similar observation was previously reported in the case of nile blue and SDS systems.16 These results suggested that NOS that moves to the micellar environments and microenvironments around the fluorophore molecule are different compared to that in the neat buffer. Also, the polarity of the micelles is less compared to that in the neat buffer and the solubility of NOS increases in SDS micelles as compared to the aqueous solvent.

Figure 3.

Figure 3

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(A) Emission spectra of NOS (50 μM) in different concentrations of SDS (0.2–10 mM). (B) Plots of variation of emission maxima of NOS in the presence of SDS.

2.1.2. Partition Coefficient Determination

The spectral changes of NOS in the presence of SDS micelles correspond to the significant penetration of NOS in the palisade layer of SDS micelles. To gain an understanding about the mechanism of accessing the NOS in SDS micelles and the understanding of the partition of NOS between the micelles and the aqueous phase, we determine the partition coefficient of aqueous and micellar systems as follows28

2.1.2. 1

where Ct, Cm, and Cw are the total molar concentration of the drug and drug concentration in the micelle and in the aqueous buffer system. [micelle] and [buffer] represent for molar concentrations of SDS micelles and water, respectively. The partition coefficient of NOS was calculated by using steady-state fluorescence data according to the following relationship.29

2.1.2. 2

where Kp is denoted as the partition coefficient of NOS to the micellar phase from the aqueous medium. I0, It, and I∞ are the fluorescence intensities of NOS in the absence of SDS, with an intermediate, and at a saturated concentration, respectively. Kp has been calculated from the slope of the (I∞ – I0)/(It – I0) versus [surfactant]−1 plot (Figure S2A).The value of Kp was obtained around 5.89 × 104. A higher Kp value showed a greater partitioning of NOS in SDS micelles.24 Moreover, this significant partitioning of NOS inside the SDS micelle suggested a strong binding between NOS and SDS micelles, leading to a stable system. This result was again confirmed by observing the binding constant between NOS and SDS. The binding constant was utilized to achieve quantitative appraisal of the strength of binding between the NOS and SDS assemblies.30 To calculate the binding constant of NOS in SDS micelles, we have employed modified Benesi–Hildebrand equation using fluorescence data. The modified Benesi–Hildebrand equation can be described as follows31

2.1.2. 3

where Kb denotes the binding constant of NOS in the micellar medium. ΔI = It – I0 and ΔImax = I∞ – I0, with I0, It, and I∞, are the fluorescence intensities of NOS in the absence of SDS, with an intermediate, and at a saturated concentration, respectively. [M] is the micellar concentration, which is expressed by the following equation32

2.1.2. 4

where [S] represents the concentration of SDS and Nagg is the aggregation number of the SDS micelle. Nagg is determined by using the standard fluorescence quenching method.33 The value of Nagg was calculated to be 281 and the consequent plot is shown in Figure S3. The value of Kb was calculated from the slope of the (I∞ – I0)/(It – I0) versus [M]−1 plot (Figure S2B). The value of Kb was obtained around 1.78 × 102 M–1. The free-energy change ΔG was also calculated by using the following relationship

2.1.2. 5

where Kb is the binding constant. The observed negative value of ΔG (−12.84 kJ mol–1) suggested that the binding process was spontaneous and energetically favorable.

2.1.3. Intercalation of ctDNA with NOS

The interactions of NOS with ctDNA have been studied separately. Figure 4 shows absorption (312 nm) and emissions spectra (467 nm) of NOS in aqueous buffer solution by adding increasing concentrations of ctDNA (2.46–47.7 μM). The absorption spectra clearly demonstrated that with the addition of ctDNA, the absorbance spectra of NOS decreased with a slight red shift (Figure 4A) of 4 nm, suggesting NOS interaction with ctDNA. Generally, hypochromism and red shifts refer to the intercalative binding mode that represents strong stacking interaction between the aromatic ring of the drug and the base pairs of ctDNA.34 This result suggested that strong intercalation occurred between NOS and base pairs of ctDNA.

Figure 4.

Figure 4

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(A) Absorption spectra of NOS (50 μM) in different concentrations of DNA (2.46–47.7 μM). (B) Emission spectra of NOS (50 μM) in different concentrations of DNA (2.46–47.7 μM).

In the emission spectral studies, when we added different concentrations of ctDNA, the fluorescence intensity of NOS gradually decreased with a minute blue shift approximately 5 nm. As shown in Figure 4B, the decrease in fluorescence intensity indicates the occurrence of an intercalative binding mode between NOS and the base pair of ctDNA. Sayed et al. also reported a similar type of results for acridine orange binding with ctDNA.18 The modified Benesi–Hildebrand equation (eq 3) is again utilized to calculate the binding constant between NOS and ctDNA interaction, where [M] denotes the ctDNA concentration. Figure S4A shows the double reciprocal plot for the NOS–ctDNA binding. The value (Kb) was found to be 1.29 × 105 M–1 at room temperature. It has been reported that the binding constants with a higher order (∼104 to 105 M–1) owing to the intercalative binding mode. Zhang et al. has reported a similar order (5 × 104) of binding constant for intercalation between ferulic acid and ctDNA.35 These results again confirmed that NOS intercalation between the base pairs of ctDNA.

To confirm the intercalative binding mode further, we carried out the ethidium bromide (EB) fluorescence displacement experiment. EB intercalates to the base pair of DNA and emits an intense fluorescence.36 Upon addition of NOS in the EB–ctDNA system, the fluorescence intensity of the EB–ctDNA system decreased (Figure S5). This decrease in the fluorescence intensity of the EB–ctDNA complex indicates that NOS displaced EB molecules and binds in the intercalative mode to ctDNA.37 The value of free energy change (ΔG = −29.17 kJ M–1) for NOS–ctDNA binding indicates that the binding process is spontaneous. From the result, it is observed that NOS exhibits a strong binding with both the SDS micelle and ctDNA independently; however, the magnitude of binding constant is higher in ctDNA compared to the SDS micellar system. These comparative binding results suggested that if ctDNA is added to the micelle-bound NOS system, NOS might prefer release from the micelle environment and binds to the ctDNA. In this prespective, we have demonstrated this assumption to be true using different spectroscopic techniques in the forthcoming sections.

2.1.4. Interaction of ctDNA with the Micelle-Loaded NOS

The interaction and binding mechanism of NOS with SDS micelles and ctDNA has already been discussed. Now, we explore the interaction of SDS-loaded NOS with ctDNA and investigate the intercalation of NOS with ctDNA from SDS micelles. As we add ctDNA to micelle-bound NOS, the absorbance spectra of NOS decreased with the slight red shift of 7 nm from 308 to 315 nm with ctDNA. The spectral modulation in Figure 5A suggested that upon addition of ctDNA, NOS experiences a different environment than that in SDS micelles (Figure 4A). The position of the absorption maximum of NOS was observed previously at 315 nm in the ctDNA medium. From Figure 1B, the parallel spectral behavior of NOS was observed with ct DNA and in the composite medium (SDS micelle + ctDNA), suggesting that NOS binds with ctDNA in the SDS micelle. Therefore, the absorption spectra result exposes that NOS prefers to intercalate with ctDNA in the composite medium (SDS micelle + ctDNA).

Figure 5.

Figure 5

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(A) Absorption spectra of SDS (10 mM)-loaded NOS (50 μM) in different concentrations of DNA (2.46–47.7 μM). (B) Emission spectra of SDS-loaded NOS (50 μM:10 mM) in different concentrations of DNA.

Emission spectral analyses were done to confirm the transfer of NOS from SDS micelles to the DNA environment. As shown in Figure 5B, it can be seen clearly that with increasing concentrations of ctDNA in micelle-loaded NOS, the emission intensity of micelle-loaded NOS decreases significantly. The quenching of NOS spectra in the composite medium (micelle + ctDNA) nearly corresponds with that of the NOS in the ctDNA medium alone (Figure 4B). Thus, the emission spectra of micelle-loaded NOS upon addition of ctDNA indicate that in the presence of ctDNA, NOS has moved from the SDS micelle to the ctDNA medium. The binding constant of micelle-loaded NOS and ctDNA was calculated using the modified Benesi–Hildebrand equation (eq 3). Figure S4B shows the double log plot for the micelle-loaded NOS–ctDNA binding. The calculated binding constant (Kb) is 3.18 × 104 M–1 at room temperature (298 K). From Table 1, it can be seen clearly that the binding constant of NOS in the composite medium is practically equivalent to the NOS–ctDNA system and comparatively much higher than that of the NOS–SDS system, that is, 1.78 × 102 M–1. Ghosh et al. reported a similar result with a cationic phenazinium dye in the lipid environment.38 This result suggested that the repositioning of NOS from the SDS micelle to ctDNA is reorganized by the contemplation of a higher binding affinity of NOS toward ctDNA in comparison to the micelle medium, leading to competitive binding.

Table 1. Binding Constant (Kb) and Gibbs Free Energy (ΔG) of NOS in Different Systems.
s.n. system Kb M–1 ΔG kJ mol–1 R2
1 NOS + SDS 1.78 × 102 ± 0.002 –12.843 0.9668
2 NOS + ctDNA 1.29 × 105 ± 0.004 –29.17 0.9824
3 micelle-loaded NOS + ctDNA 3.18 × 104 ± 0.002 –25.69 0.9968
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2.2. Probe Location Studies

For the probe location study, that is, to compare the location of the probe in the micelle and composite medium, fluorescence quenching with KI and fluorescent anisotropy studies were employed. Both studies provide the information about membrane fluidity, drug insertion, and drug location into the micelle as well as the biomolecule.28

2.2.1. Fluorescence Quenching Study with Potassium Iodide (KI)

To explore the binding location of NOS within the SDS micelle and composite medium, we employed fluorescence quenching with potassium iodide (KI) as a quencher. The fluorescence quenching study gives valuable information regarding the accessibility of the entrapped fluorophore toward the quencher and therefore it is useful in the assessment of the location of the fluorophore in different micro heterogeneous environments.5 Fluorescence quenching is a process in which reduction of emission intensity of a fluorophore occurs by a quencher.39−42 The quenching rate constants of NOS with the addition of a quencher (KI) for different systems (free NOS, NOS–SDS, NOS–ctDNA, and NOS–SDS–ctDNA) have been calculated by the Stern–Volmer equation

2.2.1. 6

where KSV is the Stern–Volmer quenching constant, I0 and I are the fluorescence intensities in the absence and presence of quencher (KI), respectively, and [Q] is the molar concentration of the quencher. The higher the magnitude of KSV, the better the quenching process, which indicates a greater degree of exposure of the fluorophore to the quencher.5Figure 6 shows the Stern–Volmer plots for KI quenching of NOS in different mediums such as aqueous buffer, ctDNA, and SDS micelles and in the composite medium. The calculated value of KSV in different systems is shown in Table 2. From Figure 6 and Table 2, the fluorescence quenching of NOS in the SDS micelle is significantly lower than that in the aqueous buffer solution. A lower KSV value of NOS in the SDS micelle indicates that NOS has occupied the hydrophobic core of the SDS micelle compared to the hydrophilic environment outside the micelle. The degree of exposure of the fluorophore toward the quencher diminishes in SDS micelles because the organized assemblies of SDS micelles were unreachable by the water-soluble quencher, which leads to a lower value of KSV. However, the values of KSV are varied in different systems. For the NOS–ctDNA system, the KSV value was also observed lesser in compression to free NOS but higher than that of NOS in the SDS micelle (Table 2). This indicates that NOS intercalates with ctDNA and is thus less accessible to the quencher. Moreover, the KSV value of NOS (in SDS micellar medium) with ctDNA was observed very close to the KSV value of NOS–ctDNA, which again suggested that NOS was released from the hydrophobic core of the micelle and favored the intercalation with ctDNA.28

Figure 6.

Figure 6

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Stern–Volmer plots for the quenching of NOS by KI ions in different systems.

Table 2. KI-Induced KSV Value of Different Systems.
s.n. system KSV M–1 R2
1 NOS + KI 1.58 × 102 ± 0.01 0.9777
2 NOS + SDS + KI 0.24 × 102 ± 0.005 0.9282
3 NOS + ctDNA + KI 0.45 × 102 ± 0.003 0.9213
4 micelle loaded NOS + ctDNA + KI 0.44 × 102 ± 0.004 0.9450
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2.2.2. Steady-State Fluorescence Anisotropy

The steady-state fluorescence anisotropy study is a powerful tool in the biophysical research that provides the location of the fluorophore in different bio or biomimetic microheterogeneous environments such as protein, DNA, and micelle lipids.30 When fluorescence probes are excited in polarized light, the emission from the probe is polarized. This degree of polarization of the emission is defined as anisotropy (r) which provides valuable information about the surrounding environments of the fluorophores.27,43 We obtained information about the size, shape, and segmental flexibility of a molecule affecting different moieties using fluorescence anisotropy. An increase in fluorescence anisotropy quantifies the rigidity of the environment surrounding the fluorophore, which is used as an indicator of the extent of rigidity imposed on the fluorophore in different micro heterogeneous mediums.19,44

In addition to the information provided by fluorescence quenching data, fluorescence anisotropy (r) values of NOS have been measured in different environments to judge its location in these mediums. Figure 7 shows the dissimilarity in the fluorescence anisotropy values of NOS in SDS micelles, ctDNA, and the composite medium, and the significant fluorescence anisotropy values are compiled in Table 3. From Figure 7, fluorescence anisotropy of NOS in the SDS micelle and ctDNA environments is considerably higher in comparison to an aqueous buffer medium (Table 3). Also, there is a gradual increase in the anisotropy value of NOS with increasing concentrations of SDS (Figure 7). These data show the imposition of some sort of rotational restriction of NOS upon binding with both the systems as the monomers of SDS are arranged more orderly. Above the cmc of SDS, the anisotropy reached a maximum and constant further. This indicates that the NOS molecule is entrapped inside the SDS micelles. Higher fluorescence anisotropy reflects the gradual increase in rigidity of the drug inside the micelle which confirms that NOS moves to a more restricted region, that is, core of the micelle. This result was in agreement with a large blue shift in the fluorescence spectra of NOS upon the addition of SDS (Figure 3A).44 In the NOS–ctDNA system, the anisotropy value increases from the value obtained in the aqueous buffer, signifying imposition of the motional restriction on NOS upon binding with ctDNA and a higher anisotropy value confirmed the intercalative mode of binding between NOS and ctDNA. When ctDNA is added in micelle-loaded NOS, the anisotropy value of NOS deceases gradually and reach 0.059 at a high ctDNA concentration (50 μM), which suggested that the environment of NOS is changed. This anisotropy value is very close to the observed NOS–ctDNA system (Table 3). The steady-state fluorescence anisotropy result again confirmed that NOS occurs in similar motional restrictions from its surrounding moiety in DNA as well as the composite medium and NOS favor the intercalation with ctDNA from SDS micelles (see Table 4).44

Figure 7.

Figure 7

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Steady-state fluorescence anisotropy(r) of NOS–SDS, NOS–DNA, and NOS–SDS–DNA.

Table 3. Steady-State Fluorescence Anisotropy of Different Systems.
s.n. system anisotropy (r)
1 NOS (50 μM) 0.031 ± 0.0008
2 NOS + SDS (10 mM) 0.096 ± 0.0026
3 NOS + DNA (50 μM) 0.056 ± 0.0016
4 NOS–SDS (10 mM) + DNA (50 μM) 0.059 ± 0.0017
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Table 4. Hydrodynamic Radius (Rh) of NOS in Different Environments.
s.n. system hydrodynamic diameter (nm)
1 SDS micelle 6.12 ± 0.29
2 ctDNA 87.66 ± 2.32
3 NOS + SDS 6.66 ± 0.91
4 NOS + ctDNA 138.4 ± 9.36
5 ctDNA + SDS 111.5 ± 10.21
6 NOS–SDS (10 mM) + ctDNA (50 μM) 152.1 ± 2.97
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2.3. Time-Resolved Fluorescence Decay

Time-resolved fluorescence decay is a very sensitive tool for studying the nature of hydration as well as the relaxation dynamics of the drug in aqueous and micellar environments and also the excited state interactions of the probe.39,45−47 This gives the information about the residence of the probe within different micro heterogeneous mediums. Herein, we observe the fluorescence lifetime of NOS in different environments to find out its replacement from micelles to the ctDNA in the composite medium. The fluorescence lifetime of NOS in SDS, ctDNA, and composite medium is shown in Figure 8 at an excitation wavelength 312 nm and the corresponding deconvoluted data are tabulated in Tables S1–S3. It can be seen from Table S1, the fluorescence lifetime of NOS (in buffer) was fitted by the biexponential function consisting shorter τ1 (1.79 ns) and longer τ2 (15.95 ns) component. This indicates the residence of the probe in a single environment.19 In micelles, ctDNA, and composite medium, NOS is fitted by a triexponential function comprising a small relative population of long components τ3, which represents the multiple locations of the probe environment differing in polarity.27,44 From Table S1 (Figure 8A), the average fluorescence lifetimes of NOS were increased significantly with the increasing concentrations of SDS, suggesting the incorporation of NOS into SDS micelles. Also, Table S1 shows drastical increase in the longer component, τ2 (15.79–19.58 ns). This enhancement of lifetimes signifies change in the environment of NOS aqueous to micellar environment. However, Figure 9A clearly shows that the τavg value from 0.2 to 1.6 mM concentration range of SDS is decreased, enhanced till 6.4 mM and then becomes constant. This result again confirmed our previous finding that NOS resides in the hydrophobic core of the micelle. In the case of NOS–ctDNA interaction, there is a slight increase in τavg (15.79–16.25 ns). Change in lifetime suggested the binding of NOS to ctDNA (Figure 8B, Table S2). When we added ctDNA in micelle-bound NOS, the decay profile of NOS was observed to be almost similar to that in ctDNA environment (Figure 8C, Table S3). Figure 8D shows the change of decay patterns of NOS in different environments (buffer, micelle, ctDNA, and composite medium). A comparative study of the τavg value of NOS in the ctDNA system and NOS in the composite medium showed the intercalation of NOS with ctDNA from SDS micelles, as in the composite medium, the τavg value was decreased on increasing the concentration of ctDNA and reach an equivalent τavg value to that of the NOS–ctDNA system (Figure 9B). Figures 8D and 9B show clearly that the lifetime of NOS in the composite medium agrees well with only the ctDNA medium, which suggested the transfer of NOS from SDS micelles to ctDNA.

Figure 8.

Figure 8

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Time-resolved fluorescence decays of NOS–SDS (A), NOS–DNA (B), and NOS–SDS–DNA (C) and a comparative study of all (D).

Figure 9.

Figure 9

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(A) Difference in variation of τavg of NOS with increased concentrations of SDS. (B) Difference in variation of τavg of NOS and micelle bound NOS with increased concentrations of ctDNA.

2.4. Time-Resolved Anisotropy

Time-resolved fluorescence anisotropy is also a very useful technique to determine the microenvironment and location of the probe in a multi-component environment. It gives an idea about structural and dynamical information of the fluorophore in an organized medium.48,49 Fluorescence decay anisotropy is directly associated with the reorientation dynamics of the excited fluorophore, and thus it is a suitable tool for the investigation of molecular dynamics and rotational relaxation and thereby for the structural information of fluorophore.50 To obtain the information about the microenvironment around the NOS in different mediums (SDS micelle, ctDNA, and composite medium), the time-resolved fluorescence anisotropy decay of NOS was performed. Figure 10 shows the time-resolved fluorescence anisotropy decay of NOS in different mediums. In the aqueous medium (buffer), the anisotropy decay of NOS is found to be single-exponential with a lifetime component of 0.499 ns. In the presence of SDS micelles, the anisotropy decay is also fitted as single-exponential, but the rotational relaxation decay time increases about 0.744 ns, which indicates that the environment of NOS was changed. The significantly enhancement in rotational relaxation decay time of NOS shows the location of the drug in a motionally constrained environment (Figure 10A). In the presence of ctDNA, the anisotropy decay of NOS is fitted biexponential with a short component and as a lower component. This interaction shows the “dip-and rise” kind of an anisotropy decay pattern, which indicates the presence of two or more populations, among which one has a short fluorescence lifetime with a fast rotational correlation time and another is a slower component.27,51 Generally, this type of an anisotropy decay profile has been observed for fluorophores withbiomolecules such as protein and DNA. In which the shorter correlation time component is credited through the free fluorophore (i.e., NOS in buffer) and the slower component is due to the fluorophores that bind with the biomolecule.27,49,52,53 When we performed time-resolved anisotropy decay of NOS in the composite medium, we obtained again biexponential function of rotational correlation time with the shorter and slower components. The pattern of the anisotropy decay profile of NOS was observed to be almost similar to that in the ctDNA environment (Figure 10B), which strongly support our previous finding, that is, NOS is released from the SDS micelle and binds with ctDNA in the composite medium.

Figure 10.

Figure 10

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(A) Time-resolved fluorescence anisotropy r(t) decay of NOS and NOS–SDS. (B) Time-resolved fluorescence anisotropy r(t) decay of NOS, NOS–DNA, and NOS–SDS–DNA.

2.5. Dynamic Light Scattering

To explore the interaction of NOS with ctDNA and the relation of NOS from SDS micelles to ctDNA in the composite medium, we have performed dynamic light scattering (DLS) as it is a well-known technique that provides valuable information about the dimension of supramolecular assemblies and biomolecules.21,49Figure S6 shows the intensity distribution of DLS profiles of ctDNA, SDS micelles, ctDNA–SDS, ctDNA–NOS, NOS–SDS, and NOS–SDS–ctDNA interaction. The DLS peak of NOS was not observed due to its smaller size. DLS spectra of SDS micelles and SDS micelle bound NOS reveal a quite monomodal distribution with an average diameter of 6.12 and 7.41 nm, respectively (Figure S6A,B). The NOS–SDS medium shows much broadened distribution with a higher diameter relative to the SDS micelle, which indicates the incorporation of NOS to SDS micelles.29 The DLS profile of native ctDNA shows two peaks (Figure S6C), where the second larger peak with hydrodynamic diameters of 87.66 nm corresponds to the hydrodynamic diameter of native ctDNA.54 In the ctDNA–SDS system, two peaks were observed with hydrodynamic diameters of 6.12 and 111.5 nm (Figure S6D), wherein the first peak corresponds to the SDS micelle and the second peak corresponds to ctDNA. As we added NOS in the ctDNA medium, a large peak at 138.4 nm was observed (Figure S6E). A slightly enhanced hydrodynamic radius with a small shift toward a higher dimension indicates NOS intercalation with ctDNA which results in an incremental increase in the diameter of NOS. In the composite medium, we observed multiple peaks, which represents more than one type molecule found in medium29 (Figure S6F). From Figure S6F, the first peak corresponds to free micelles, the second peak for SDS bound NOS, and the third large peak is due to the intercalated ctDNA with NOS. The DLS result again confirms our observation by other techniques that in the composite medium NOS is released from the SDS micelle and intercalated with ctDNA.

2.6. Effect of SDS Micelles on the Structure of ctDNA

It is very crucial to determine the effect of SDS micelles on the ctDNA structure; hence we observe the structural aspect of ctDNA using CD analysis below and above the cmc of SDS. Figure 11A shows the CD spectra of ctDNA with two concentration of SDS, one is below cmc (4 mM) and another is above cmc (10 mM). The CD spectrum of ctDNA shows a two characteristic peaks attributed to the right-handed helicity: a positive peak at ∼275 nm and a negative peak at ∼245 nm.55 There is no significant change in the CD spectrum of ctDNA observed at these concentrations of SDS, which suggested that the SDS micelle does not perturb the structure of ctDNA. Patra et al. also reported that SDS micelles do not give the structure effect on ctDNA.56 This result supported that we can use SDS micellar systems as the carrier of drugs for their safe delivery to the target, DNA.

Figure 11.

Figure 11

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(A)CD spectra of ctDNA (100 μM) in the presence of different concentrations of SDS. (B)CD spectra of ctDNA (100 μM) in the presence of NOS (50 μM) and SDS micelle-loaded NOS.

Further, we analyzed the CD spectrum of NOS in different environments to determine the relocation of NOS from SDS micelle to ctDNA in the composite medium. NOS has a chiral atom, and so it exhibits its own CD spectrum with two negative peaks at 218 and 238 nm (Figure S7). As we introduced SDS to the NOS, the peak at 218 nm disappeared, which indicated that SDS induces structural changes in NOS which may be due to the hydrophobic environment created by SDS micelles and NOS resides in the hydrophobic core of the SDS micelle (Figure S7). Further upon addition of NOS in ctDNA, it induces a strong structural change in ctDNA as the intensity of both the peaks were changed largely (Figure 11B). The strong change in both the peaks is attributed to the imposed asymmetry on the probe because of its intercalation within the DNA base pairs. Induced CD spectra of DNA binders originate because of the coupling of the electric transition dipoles of the binder molecule and DNA bases within the asymmetric DNA environment.57 In the SDS micellar medium, we observed similar results as those observed for the NOS–ctDNA interaction. The structural peak of ctDNA changes in the composite medium, which indicates the relocation of NOS from SDS micelles to ctDNA and intercalate to ctDNA (Figure 11B). Therefore, the result suggested that in the presence of ctDNA, NOS binds to ctDNA and releases from the micelle.

2.7. Molecular Docking Study

We utilized the molecular docking technique to determine the binding location of a small molecule within the microheterogeneous assembly as well as the biomolecule.58−60 Herein, we have carried out molecular docking technique through AutoDock software to assess the probable location and binding mode of NOS in SDS micelles and ctDNA. We performed 100 docking run of NOS with SDS micelles built in a micelle maker server and DNA duplex of sequence d(CGCGAATTCGCG)2 dodecamer (PDB ID: 1BNA), and the most energetically favorable conformation was utilized for the docking analysis. NOS shows strong binding with both SDS micelles (−11.02 kcal) and ctDNA (−7.0 kcal), which is comparable to our experimental result. As shown clearly in Figure 12A, NOS lies between the base pairs of ctDNA in the binding site of the G–C region, which suggests that the drugs bind to ctDNA through intercalative binding. Figure 12B shows that NOS binds through three base pairs G–C, C–G, and A–T of ctDNA with two hydrogen bonds between NOS and G4, A5 residue of the ctDNA. However, Figure 12C shows binding between NOS and SDS micelles, which clearly indicates that NOS resides in the core of the micelle through hydrophobic interactions. The molecular docking results suggested the binding of NOS with SDS micelles and ctDNA, which shows a mutual coherence between computational and spectroscopic techniques.

Figure 12.

Figure 12

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(A) Molecular docked structures NOS with ctDNA. (B)Surrounding nucleotide residue of ctDNA within 5 Å from docked NOS. (C) Molecular docked structures NOS with SDS micelles.

3. Conclusions

In this work, we have explored the intercalation of an anticancer drug, NOS, to ctDNA through an anionic SDS micelle by using different spectroscopy techniques such as absorption, steady-state, time-resolved fluorescence, CD spectroscopy and DLS measurements. The binding affinities of NOS–SDS micelles and NOS–ctDNA are 1.78 × 102 and 1.29 × 105 M–1, respectively. Higher binding constant signify the relocation of NOS from SDS micelles to ctDNA. The locations of NOS in different environments were determined by fluorescence quenching and steady-state fluorescence anisotropy, which confirms the transfer of NOS. Time-resolved fluorescence, time resolved fluorescence anisotropy, and CD studies substantiate that in the composite medium, NOS remains intercalated within the DNA base pairs and are released from SDS micelles. Additionally, our molecular docking results again confirmed that NOS resides into the core of the micelle.

4. Experimental Section

4.1. Materials

ct-DNA, NOS (98%), SDS, and phosphate salts (sodium monophosphate and biphosphate) were obtained from Sigma-Aldrich. The stock solution of ctDNA was prepared by dissolving in phosphate buffer (10 mM, pH 7.0) and stored in −20 °C, as described in the literature.18 The ctDNA concentration was determined spectrophotometrically using extinction coefficients (ε) 6600 M–1 cm–1 at 260 nm and the purity of ctDNA was confirmed by observing absorbance ratio (A260 nm/A280 nm) which is observed between the range of 1.8–1.9.19 NOS solution was prepared in the phosphate buffer. The freshly prepared micellar solution of SDS was utilized after confirming its cmc by using a Langmuir tensiometer (Kibron, Helsinki, Finland). The cmc of SDS in water at room temperature was observed at 8.2 mM, which is matches well with reported values.5 Ultrapure water was used throughout the experiments obtained from a Millipore water purification system. The buffer solution was filtered through Millipore filters of 0.22 μm pore size. All other reagents were of analytical reagent grade and used without further purification.

4.2. Instrumentation

An Analytik Jena Specord-210 spectrophotometer and a Cary Eclipse fluorescence spectrometer (Varian, USA) were used to measure the absorption and emission spectra. The steady-state anisotropy (r) was measured on the same spectrofluorometer equipped with a manual monopolarizer by quantifying the fluorescence intensities with the excitation polarizer oriented vertically and the emission polarizer oriented vertically (IVV) and horizontally (IVH), respectively, using following equation

4.2. 7

The grating factor (G) is calculated by the following relation

4.2. 8

The lifetime decay was observed on a time-correlated single-photon counting spectrometer (Horiba, Jobin Yvon, IBH Ltd., Glasgow, UK). Moreover, the time resolved fluorescence anisotropy decays were also carried out with the same instrument using a motorized polarizer. The emission intensity was collected at parallel (I||) and perpendicular (I⊥) polarization decay with a certain peak difference. The same software was utilized for the analysis of the anisotropy data. The time-resolved anisotropy decay function r(t) was described as the following relation

4.2. 9

where r(t) represents the rotational relaxation correlation function. I||(t) and I⊥(t) are fluorescence decay for parallel and perpendicular polarization, respectively. G is the grating factor, which is described by eq 4

4.2. 10

For size measurements, the DLS study was done on Malvern Zetasizer Nano ZS90. The conformational changes in ctDNA structure were observed with a Jasco-715 spectropolarimeter attached with a temperature controlling unit for uniform temperature. The molecular docking study was also carried out on Autodock 4.2 software. Further details of methodology has been described in our earlier publication15,20,21 and in the Supporting Information. For NOS–ctDNA binding, we have used a fixed concentration of NOS (50 μM) with varying concentrations of ctDNA (2.46–47.7 μM). Also, for the NOS–SDS micellar interaction, fix concentration of NOS (50 μM) and different concentrations of SDS (0.2–10 mM) below and above cmc were utilized. For the NOS–SDS–ctDNA system, fix concentrations of NOS and SDS complex (50 μM:10 mM) and different concentrations of ctDNA (2.46–47.7 μM) were utilized.

Acknowledgments

Dr. R.P. greatly acknowledges the financial support from Science and Engineering Research Board (EEQ/2016/000339) New Delhi, India. N.M. is also thankful to the ICMR, New Delhi, India, for providing a research grant (file Gen.452/Estt./R.O./2018).

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.9b01543.

  • Time-resolved fluorescence, dynamic light scattering and UV spectroscopy (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao9b01543_si_001.pdf (491.2KB, pdf)

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