Ionic Liquid Green Assembly-Mediated Migration of Piperine from Calf-Thymus DNA: A New Possibility of the Tunable Drug Delivery System.

Biocompatible surface-active ionic liquid (SAIL) was used first to study the deintercalation process of a well-known natural compound piperine (PIP) as an anticancer drug, obtained from PIP-calf thymus DNA (ctDNA) complex under controlled experimental conditions. In this study, we have been exploring the interaction of PIP in SAIL (1-butyl-3-methylimidazolium octyl sulfate ionic liquid ([C4mim][C8OSO3])), ctDNA, and deintercalation of PIP from the PIP-ctDNA complex through SAIL micelle using various spectroscopic techniques. Absorption, emission, and lifetime decay measurements provide strong evidence of the relocation of PIP molecules from ctDNA to SAIL micelle. Fluorescence quenching and steady-state fluorescence anisotropy were employed to examine the exact location of PIP in different media. Moreover, the surface tension technique was also employed to confirm the release of PIP molecules from the PIP-ctDNA complex in the presence of SAIL. Circular dichroism analysis suggested that SAIL micelle does not perturb the ctDNA structure, which supported the fact that SAIL micelle can be used as a safe vehicle for PIP. Overall, the study highlighted a novel strategy for deintercalation of drug using SAIL because the release of the drug can be controlled over a period by varying the concentration and composition of the SAIL.

Introduction

Deoxyribonucleic acid (DNA) is a chief target for many anticancer drugs because of this drug–DNA interaction that provides valuable information in the cancer research. Drug–DNA interaction gives an idea about the three-dimensional shape of the complex and the binding mechanism, that is, whether it is a covalent or noncovalent binding. It is well-known that the noncovalent binding of drug–DNA interaction are classified into three broad classes: (1) intercalation, (2) groove binding, and (3) electrostatic interaction. In the intercalation, the drug binds to DNA between its adjacent base pairs of strand, whereas in groove binding, the drugs fit into the minor or major groove of DNA.[1,2] The release of the drug from the DNA was also an important fact in pharmacology research. On this prospect from the last few years, researchers are focused on the deintercalation processes through different carriers.[3−5]

Deintercalation is the process of removal of bound ligands or drugs from the biomolecule (DNA or protein) with the help of a carrier (such as micelles and liposome). The micelles are the simplest structure that easily modulate the drug DNA binding equilibrium. These nanostructures provide a hydrophobic environment to the drug by which it deintercalates from DNA and absorbs into the hydrophobic core of micelles without disturbing the DNA structure.[6,7] Westerlund et al. have reported the dissociation of cationic DNA binding ligands in pre- and postmicellar concentration ranges of the monomeric surfactant.[5] Also, Chakraborty and co-workers have reported the deintercalation processes of doxorubicin from DNA through different biocompatible liposomes.[4] Patra et al. have also carried out the thermodynamic study about the deintercalation process of the cationic dyes phenosafranine (PSF) from DNA using SDS micelle.[3] From the vast literature survey, there is still no report of deintercalation processes using ionic liquid (ILs) micelles. ILs are novel greener solvent with many fascinating properties. From the last few decays, research studies explored the IL as a drug carrier in life sciences and medical field because of its highly tunable nature, low toxicity, greenness, low bioaccumulation, bioavailability, and selective catalytic behavior.[8−11] Herein, we utilized the surface-active IL (SAIL) micelle for deintercalation of the drug from the calf thymus DNA (ctDNA).

It was also reported that the rate of deintercalation depends on the hydrophobic chain length of the carrier. Therefore, in this work, we have utilized more hydrophobic anionic SAIL, that is, 1-butyl-3-methylimidazolium octyl sulfate ([C4mim][C8SO4]), instead of surfactants or liposome because the presence of long alkyl chain in its anionic moiety provides a special character; as a result, they behave as surfactants. Moreover, it was also reported that ILs are less toxic and more greener than surfactants, which make them safe drug carriers than surfactants.[12,13] The critical micelle concentration (cmc) of ([C4mim][C8SO4]) is 31.35 mM, which was confirmed by Langmuir microtensiometer.[14] Recently, researchers have a great interest to study the photophysical and chemical properties of many drugs or natural product in the SAIL and explore SAIL-containing microemulsions and micelles.[15−17] From the overall viewpoint, we choose the [C4mim][C8SO4] because of its modest biodegradation properties over other ILs.[18] However, SAILs have found to increase the drug permeability of the biological membranes. Owing to the small size of the micelle and good stability of the drug in micelle, the micelle can act as good drug carriers with advantages over other drug carriers.[10,19] Moreover, the micelle enhances drug bootability and reduces the loss, degradation, and hazardous side effect of the drug. This is all possible because micelles lessen the contact of the drug molecule with enzyme and other body fluids when compared to the free drug molecule.[10,20] The experiment by Rangel-Yagui et al.[21] showed the solubility of the drug molecules in the micellar system because of the association between the drug and the surfactant micelle. Similarly, Mahajan et al.[22] have studied the extent of binding of drug (dopamine hydrochloride and acetylcholine chloride) with SAILs and compared with conventional surfactant TTAB. They suggested that drugs interact in the outer portion of the SAIL micelle and surfactant, but SAILs act as better carriers for drug than surfactants. From the literature, it was observed that SAILs acts as a better drug carrier compared to conventional cationic surfactants.[10,16,22−26]

In this work, we have shown the intercalation of piperine (PIP) with ctDNA and followed by deintercalation in the presence of SAIL micelle (Scheme ). PIP (1-piperoylpiperidine) is the major bioactive alkaloid phytochemical extracted from the fruit and the root of black pepper (Piper nigrum L.) and long pepper (Piper longum L.) plants from the Piperaceae family[27] (Scheme ). It has been reported recently that PIP controls and reduces the progression of tumor cell growth and induces apoptosis.[28] Lai et al. reported that PIP suppressed the tumor growth and metastasis of 4T1 cell of breast cancer in the mouse model.[29] It was also reported that PIP inhibits the development of prostate cancer cells mediated through decreased expression of the nuclear factor-κB (NF-κB) and phosphorylated STAT-3 in LNCaP cells.[30] PIP also has the ability to bind with the G-quadruplex DNA structure, which makes it more significant as a chemotherapeutic agent for cancers; it also provides deviations in the DNA metabolism.[31]

Scheme 2

Systematic Representation of Deintercalation of PIP from ctDNA to Micelle

Scheme 1

Structure of PIP

We have divided our present work into three sections (Scheme ). The first section explains about the encapsulation of PIP in a micellar environment formed by SAIL. The second section deals with the interaction of PIP with ctDNA, followed by the third section which deals with the deintercalation of PIP from the PIP–DNA complex in SAIL by using different spectroscopic techniques and surface tension method.

Results and Discussion

Absorption and Emission Spectral Study

The absorption spectra of PIP observed three absorption bands at 250 and 342 nm with a small shoulder of 311 nm in an aqueous buffer solution (Figure S1A), and the emission spectra of PIP was found to be a single strong peak at 486 nm in an aqueous buffer solution (Figure S1B). To determine the behavior and encapsulation of the PIP in SAIL micelle microenvironment, the absorption and emission spectroscopy was utilized.[3]Figure demonstrates the absorption spectra of PIP in the absence and presence of different concentrations of SAIL (2–35 mM). After the addition of SAIL, the intensity of all absorbance bands steadily increased with a minor blueshift (342–335 nm) (Figure A). Figure B represents the relative change in the absorbance of PIP at 342 nm and a maxima absorbance of the PIP against the pre- to postmicellar concentration of SAIL. The blueshift is an indicator that PIP molecules go to more hydrophobic region, which signifies an environment change from bulk aqueous to SAIL micellar region.[16] Conclusively, as the environment drifts from monomeric SAIL to micellar SAIL, the absorption spectrum shows a large hyperchromicity. From Figure B initially, at a lower concentration of SAIL (2 mM), the decrease in the absorbance of the PIP is because of the involvement of electrostatic interaction.[32] After this, as the concentration of the SAIL increases, a blueshift with hyperchromicity was observed in the absorbance of PIP which suggested that PIP was entrapped into the core of the micelle because of hydrophobic–hydrophobic interaction.[32] Further, a relative change in the absorbance and alteration in maxima absorbance of PIP recommended that PIP goes from polar to nonpolar environment and is situated inside the hydrodynamic sphere of the SAIL micelle.

Figure 1

(A) Absorption spectra of PIP (50 μM) in different concentrations of SAIL (2–35 mM) and (B) plots of variation of relative absorbance (red) and absorbance maxima (blue) of PIP in the presence of SAIL.

The emission spectrum of PIP shows a strong single peak at 486 nm when exited at 342 nm[33] (Figure S1B). SAIL also shows emission spectra at 415 nm at its higher concentration; however, it does not obstruct the emission peak of PIP (Figure S2A,B). Figure A shows that on the addition of SAIL (pre-cmc to post-cmc concentration), the intensity of PIP steadily increases with a large blueshift. Many-fold enhancement of the fluorescence relative intensity and the large blueshift about 22 nm (486–464 nm) show that PIP interacts with SAIL and alters the microenvironment of PIP through the relocation of fluorophore molecule from a high-polarity environment (buffer) to a low polar region (the hydrocarbon core of the SAIL micelle) (Figure B). This result also favored a stronger binding between PIP and SAIL and a possible intake of PIP molecules into the SAIL micelle. A similar observation was previously reported in cases of PSF and SDS systems.[3] The overall absorption and emission spectroscopy result suggested that the microenvironment around the fluorophore molecule is different when compared to its native environment, and PIP is introduced to the micellar region of SAIL. The emission spectral change of PIP in the presence of SAIL was further utilized to determine the penetration of PIP in the SAIL micelle. To support our absorption and emission results related to the penetration of PIP into the SAIL micelle, the partition coefficient was also determined using the following relationship[34]where Kp is the partition coefficient of PIP from aqueous to micellar phase. I0, I, and I∞ are the fluorescence intensities of PIP in the absence of SAIL, an intermediate, saturated concentration, respectively. The value of Kp was calculated from the slope of the (I∞ – I0)/(I – I0) versus [SAIL]−1 plot (Figure S3). The value of Kp was obtained around 3.9 × 103. The high magnitude of Kp for PIP and SAIL micelle system suggested partitioning of the PIP in the SAIL micelle.[16] Moreover, the high value of Kp suggested that there is a favorable binding between PIP and SAIL micelle, which is a stable system.

Figure 2

(A) Emission spectra of PIP (50 μM) in different concentrations of SAIL (2–35 mM) and (B) plots of variation of emission maxima (red) and relative fluorescence intensity (blue) of PIP in the presence of SAIL.

The absorption and emission profile of PIP with an increasing concentration of ctDNA (0.22–1.25 μM) is depicted in Figure . The absorption spectra show that the addition of ctDNA to PIP solution causes substantial decrease in the absorbance spectra of PIP without any considerable shift (Figure A). It is a previous report and generally accepted information that groove binding corresponds to the insignificant or minor shift in the absorbance maxima, whereas a large shift is found in the intercalation process when the ligand binds to DNA with a base stack.[35,36] This phenomenon suggested that PIP binds with ctDNA through groove binding.[33] In the emission spectral studies (Figure B), the fluorescence intensity of PIP was decreased gradually with the addition of different concentrations of ctDNA (0.22–1.29 mM) without any shift in maxima emission. The extreme amount of quenching was observed at the maximum concentration of ctDNA (1.29 mM), indicating that maximum PIP molecule binds to ctDNA and a negligible quantity of unbounded PIP was leftover in the aqueous solution. Das et al. also reported a similar type of results for PSF binding with ctDNA.[4] To confirm the exact binding mechanism of PIP to ctDNA, we performed the ethidium bromide (EB) fluorescence displacement experiment. EB is a well-known intercalating agent, and it binds between the base pairs of DNA, which cause enhancement of the fluorescence intensity.[37] On the addition of PIP (up to 50 μM) in to the EB–ctDNA system, the fluorescence intensity of the EB–ctDNA system was decreased insignificantly (Figure S4). Small reductions in fluorescence intensity of the EB–ctDNA complex signify that PIP does not displace EB molecules from the ctDNA. This result again confirmed the groove binding between PIP and ctDNA.[38] The Stern–Volmer quenching constant (Ksv) was also determined from the fluorescence data using Stern–Volmer equation[39,40] and was found to be 3.25 × 103 M–1 (Figure S5). This result exposed that with the addition of ctDNA, the fluorescence intensity was dropped rapidly and ctDNA was bound to PIP.

Figure 3

(A) Absorption spectra of PIP (50 μM) in different concentrations of ctDNA (0.22–1.29 mM) and (B) emission spectra of PIP (50 μM) in different concentrations of ctDNA (0.22–1.29 mM).

The deintercalation of PIP from PIP–ctDNA complex to SAIL micelle has been observed spectrophotometrically. The absorption and emission profiles of PIP bound ctDNA (1:25 stoichiometric ratio at maximum binding, as previously seen from the PIP–ctDNA binding study) with a rising concentration of SAIL (2–35 mM) are shown in Figure . As SAIL was added to the PIP–ctDNA complex, the absorbance spectra of PIP increased with a blueshift from 340 nm in ctDNA to 334 nm in micellar medium. Enhancement in absorbance spectra and blueshift at cmc concentration showed that the microenvironment of PIP changes, that is, PIP migrates from ctDNA to SAIL micelle (Figure A). The position of the absorption maximum of the PIP was observed (Figure A) at 335 nm in the SAIL micelle medium. The overall absorbance study of PIP in SAIL (Figure B) and PIP–ctDNA complex in SAIL (Figure B) was suggested that PIP relocated from ctDNA to the micellar environment of the SAIL.[41] Further, the deintercalation phenomenon, that is, relocation of PIP from ctDNA to SAIL micelle, was confirmed by emission spectra. As shown in Figure A, the fluorescence intensity of PIP–ctDNA complex with increasing concentrations of SAIL was increased with a considerable blueshift about 20 nm. This large shift along with the evident increase in fluorescence intensity indicates that PIP resides into the core of SAIL micelle (Figure B). Figure again shows the relocation of PIP from ctDNA to SAIL micelle. The overall absorbance and emission spectroscopy results confirmed that PIP was encapsulated in SAIL micelle and showed groove binding with ctDNA. Further, it was confirmed that as we increase SAIL concentration (above cmc) with ctDNA–PIP complex, PIP dissociates from ctDNA to micellar environment of SAIL. On this viewpoint, we will again confirm this assumption through other techniques in the forthcoming sections.

Figure 4

(A) Absorption spectra of PIP-bounded ctDNA (1:25) in different concentrations of SAIL (2–35 mM) and (B) plots of variation of relative absorbance (red) and absorbance maxima (blue) of PIP–ctDNA complex in the presence of SAIL.

Figure 5

(A) Emission spectra of PIP–ctDNA complex (1:25) in different concentrations of SAIL (2–35 mM) and (B) plots of variation of relative emission (red) and emission maxima (blue) of PIP in the presence of SAIL.

Figure 6

Comparative emission spectra of PIP (50 μM) in different systems.

Isothermal Titration Calorimetry

We have utilized isothermal titration calorimetry (ITC) for observing the thermodynamic and binding parameters of PIP in SAIL and ctDNA, and also for deintercalation of PIP from ctDNA to SAIL micelle. The ITC profiles of the titration of SAIL in PIP and PIP–ctDNA complex, and PIP with ctDNA are shown in Figure , and the corresponding thermodynamic and binding parameters are listed in Table . Figure A and Table showed clearly that the migration of PIP to SAIL micelle is an enthalpy-driven process (ΔH0 = −5.57 kcal mol–1) with negative entropy change (TΔS0 = −2.98 kcal mol–1). The free-energy change (ΔG0) was also calculated from the following relationship

Figure 7

ITC profiles for the (A) PIP + SAIL interaction, (B) PIP + ctDNA complex formation, and (C) PIP–ctDNA (1:25) complex with SAIL.

Table 1

Binding Constant (Kb) and Thermodynamic Parameter of PIP in Different System Drive from ITC

s. no.	system	Kb M–1	ΔH (kcal mol–1)	TΔS (kcal mol–1)	ΔG (kcal mol–1)
1	PIP + SAIL	78.1 ± 3.6	–5.57 ± 0.25	–2.98	–2.59
2	PIP + ctDNA	10.1 × 104 ± 40	–71.80 ± 1.44	–64.96	–6.84
3	PIP–ctDNA + SAIL	105 ± 8.5	–0.35 ± 0.07	2.40	–2.75

The negative values of ΔG0 (−2.59 kcal mol–1) shows spontaneity in the binding process. The large negative value of enthalpy shows that the migration of PIP to SAIL micelle involved the disruption of water structure at micellar surface which provides a more hydrophobic environment to PIP than its previous state.[3,32] This result is in good agreement with our spectroscopic results. The binding of PIP with ctDNA was also governed through the exothermic process (ΔH0 = −71.80 kcal mol–1) with a large negative entropy contribution (TΔS0 = −64.96 kcal mol–1). The calculated ΔG0 value (−6.84 kcal mol–1) further confirms that the binding process was spontaneous (Figure B and Table ). Moreover, the binding constant (Kb) values for PIP–ctDNA binding was also observed and listed in Table and found to be very close to the previously reported work, which confirm the groove binding of PIP with ctDNA.[33] In continuation to this, Figure C shows the deintercalation of PIP from ctDNA to SAIL micelle. The less negative value of enthalpy change (ΔH0 = −0.35 kcal mol–1) was observed for PIP–ctDNA complex in the presence of SAIL micelles with a positive entropic contribution (TΔS0 = 2.40 kcal mol–1). The positive entropic contribution showed that the migration of PIP to SAIL micelle from ctDNA is much favorable. The Gibbs free energy (ΔG0 = −2.75 kcal mol–1) was observed negative which suggested that the deintercalation process was spontaneous. These thermodynamic values are complement to the values observed for the PIP-SAIL system. Also, the value of Kb for deintercalation process (105 M–1) was observed very close to the Kb value of PIP–SAIL system (78.1 M–1), which authenticates our discussion about the relocation of PIP from ctDNA to SAIL micelle. However, the value of Kb for PIP–ctDNA system was very high (10.1 × 104). This binding result from ITC again suggested that in composite systems, PIP favors to binds in the SAIL micelle and releases from the ctDNA.[3]

Fluorescence Quenching Study

Fluorescence quenching is a general phenomenon wherein emission intensity of fluorophore is reduced by a quencher.[42−45] The fluorescence quenching method using potassium iodide (KI) as a quencher have been performed to determine the location of PIP in different system. KI-induced quenching method provides an idea about the accessibility of the entrapped or bound fluorophore toward the quencher; as a result, it provides information about the location of the fluorophore in microheterogeneous environments. The quenching rate constants of PIP on the addition of quencher (KI) for different system have been calculated by the Stern–Volmer equation[46,47]where Ksv is the Stern–Volmer quenching constant, I0 and I are the fluorescence intensities in the absence and presence of the quencher (KI), and [Q] is the molar concentration of the quencher. The higher the magnitude of Ksv, the higher will be the quenching efficiency of the quencher, which recommends a greater degree of exposure of the fluorophore to the quencher. Figure represents the Stern–Volmer plots for the KI induced quenching of PIP in different medium such as buffer, ctDNA, SAIL micelle, and PIP–ctDNA complex in the SAIL micelle. The value of Ksv of PIP in different systems is listed in Table . It is clearly seen in Figure and Table that the fluorescence quenching of PIP in SAIL micelle is significantly lower than that of PIP in the buffer solution. The lower Ksv value of PIP in SAIL micelle suggested that the environment of PIP changes when compared to the buffer and PIP residing into the hydrophobic core of the SAIL micelle. The degree of exposure of the fluorophore toward the quencher diminishes in the SAIL micelle, and because of these organized assemblies, the fluorophore was unreachable to the water-soluble quencher (KI) and therefore, there was drop in its Ksv values. Moreover, the Ksv values of PIP–ctDNA complex and PIP in SAIL micelle were observed to be lower than that of free PIP (buffer). The lower Ksv value indicates that PIP shows groove binding with ctDNA. Table showed that SAIL was added in PIP–ctDNA complex, and the Ksv value of complex was increased and very close to PIP in the micellar system. Thus, KI induce fluorescence quenching study, confirming that PIP binds with ctDNA, and in micellar environment, it relocates to SAIL micelle from ctDNA.[48]

Figure 8

Stern–Volmer plots for the quenching of PIP by KI ions in different system.

Table 2

KI-Induced Ksv Value of Different Systems

s. no.	System	Ksv M–1	R2
1	PIP + KI	2.47 × 103 ± 0.0011	0.9777
2	PIP + ctDNA + KI	3.9 × 102 ± 0.0005	0.9282
3	PIP + SAIL + KI	1.67 × 103 ± 0.0010	0.9213
4	ctDNA bounded PIP + SAIL + KI	1.44 × 103 ± 0.0012	0.9450

Steady-State Fluorescence Anisotropy

To determine the exact location of the fluorophore in different biological and microheterogeneous mediums (such as protein, nucleic acid, lipid, micelle, and vesicle), we employed steady-state fluorescence anisotropy.[49] When the fluorophore of molecule was excited in polarized light, the emission from the fluorophore was polarized. This degree of polarization of the emission is defined as anisotropy (r), which provides a precious idea about the surrounding environments of the fluorophores.[46,50] The steady-state fluorescence anisotropy gives an idea about the size, shape, and segmental flexibility of a molecule affect in various moieties.

Fluorescence anisotropy (r) value of PIP was determined in different mediums to find out the location of PIP in these mediums. Figure shows the fluorescence anisotropy values of PIP in different medium. As shown in Figure , the fluorescence anisotropy values of PIP in all environment were lower when compared to those in a buffer medium (r = 0.330), which indicate the decrease in rigidity at the microenvironment around the PIP upon binding with ctDNA and SAIL. In the case of the PIP–SAIL system, the fluorescence anisotropy value of PIP was decreased with the increasing concentration of SAIL (Figure inset). With the increasing concentration of SAIL, the monomers of SAIL are arranged in a systematic order and restrict the rotational motion of fluorophore. After reaching micellar concentration, the anisotropy value of PIP was constant (r = 0.261) which indicates that the PIP molecule goes inside a more restricted region of the micelle. Fluorescence anisotropy result was also conformed to a large blueshift in the fluorescence spectra. In PIP–ctDNA system, the fluorescence anisotropy value of PIP gradually decreased with the addition of ctDNA which goes upto 0.280. The lower fluorescence anisotropy value indicates that PIP binds with ctDNA through groove binding. General observation shows that in the case of intercalative binding, the fluorescence anisotropy of fluorophores are much higher, that is, more than 0.30.[51,52] Sahoo et al. also reported the lower anisotropy value (r = 0.20) for (dimethylamino)styryl)-1-methylpyridinium iodide with ctDNA and showed the groove binding between them.[35] Therefore, the steady-state fluorescence anisotropy results again confirm PIP binds with the ctDNA through groove binding. As SAIL is added into the PIP–ctDNA complex, the anisotropy value of PIP deceases gradually and reaches 0.21 above the cmc concentration; this indicates the change in the environment of PIP. The observed fluorescence anisotropy value of the PIP–ctDNA complex in SAIL environment is close to the value for PIP in SAIL medium, which again confirmed that PIP is released from ctDNA and is incorporated into to the SAIL micelle.[49]

Figure 9

Steady-state fluorescence anisotropy (r) of PIP in different systems.

Time-Resolved Fluorescence Decay

Time-resolved fluorescence decay serve as a sensitive tool to investigate the nature of hydration and the relaxation dynamics of the probe in different microheterogeneous environments and excited-state interactions of the probe.[53−55] We monitored the fluorescence lifetime of PIP in different environments (SAIL micelle and ctDNA) to find out deintercalation of PIP from ctDNA to the SAIL micelle. The fluorescence lifetime of PIP in SAIL, ctDNA, and PIP–ctDNA complex in SAIL medium are shown in Figure at an excitation wavelength of 342 nm, and the corresponding deconvoluted data are listed in Tables , 4, and 5. The fluorescence lifetime of PIP in buffer and with ct DNA showed biexponential decay. PIP in buffer medium was composed of longer τ1 (3.632 ns) and shorter τ2 (0.007 ns) components where the shorter component shows 100% amplitude. This indicates the residence of the probe in a single environment.[56] In the buffer solution, mainly, the biexponential decay is found because of the existence of different hydrogen bonded molecules encircled with water molecules.[57] Moreover, the origin of bi- or multiexponential decay of fluorophore may arise because of different polarity environments in micro-heterogeneous medium.[36,58] With increasing concentrations of the ctDNA, the contribution of longer component takes place in the major role and the average lifetime (τavg) increases up to 2.065 nm (Table ). The enhancement in lifetime suggested the binding of the PIP to ctDNA. In SAIL environment, PIP are fitted by triexponential function comprising a small relative population of long components τ3, which represents multiple locations of the probe environment differing in polarity.[46,51] In the PIP–SAIL interaction, with the increasing concentrations of SAIL, the average fluorescence lifetime of PIP was increased drastically up to 6.560 ns, lowering the polarity of surrounding PIP suggesting the incorporation of PIP in SAIL micelle (Table ). Also, we added SAIL in PIP–ctDNA complex and the average lifetime increased from 2.065 to 6.454 ns (Table ), which is almost same as PIP alone in the SAIL environment (Table ). Moreover, Figure clearly showed the comparative study of τavg value of PIP and PIP–ctDNA complex in SAIL environment which evidently indicated deintercalation of PIP from PIP–ctDNA complex to SAIL micelle. The τavg value of PIP–ctDNA complex was increased on increasing the concentration of SAIL and reached equivalent to τavg value as that of the PIP–SAIL system (Figure ). Almost identical lifetime average values of both environments (PIP in SAIL and PIP–ctDNA complex in SAIL) suggested that PIP released from ctDNA and entrapped in SAIL micelle. Figure D shows the change of decay patterns of PIP in different environments (buffer, micelle, ctDNA, and composite medium). As shown in the figure, the lifetime of PIP–ctDNA in SAIL environment agrees well with PIP in the SAIL medium, which suggested deintercalation of PIP from ctDNA to SAIL micelle.

Figure 10

(A) Time-resolved fluorescence decays of PIP in SAIL, (B) ctDNA, (C) PIP–ctDNA complex in SAIL, and (D) comparative plot for all systems.

Table 3

Fluorescence Decay of PIP at Different Concentrations of ctDNA

conc. (mM)	τ1a (ns)	τ2a (ns)	α1	α2	χ2	⟨τ⟩a
0	3.632	0.007	0	100	1.109	0.007
0.220	0.619	3.190	99.090	0.910	1.132	0.735
0.580	0.112	3.242	97.150	2.850	1.236	1.549
0.860	0.107	3.588	96.160	3.840	1.386	2.099
1.100	0.105	3.319	94.450	5.550	1.435	2.192
1.250	0.142	3.314	93.810	6.190	1.471	2.065

The mean error estimated for the lifetime parameters: τ1 is ±0.03 ns, τ2 is ±0.2 ns, and ⟨τ⟩ is ±0.12.

Table 4

Fluorescence Decay of PIP at Pre- to Postmicellar Concentrations of SAIL

conc. (mM)	τ1a (ns)	τ2a (ns)	τ3a (ns)	α1	α2	α3	χ2	⟨τ⟩a
0	3.632	0.007		0	100		1.109	0.007
5	1.723	0.013	6.795	0.010	99.970	0.020	1.101	0.668
11	2.046	7.858	0.060	5.370	5.450	89.180	1.122	6.074
21	1.640	7.341	0.006	5.550	5.620	88.830	1.296	6.247
31	1.742	7.443	0.006	5.320	6.140	88.540	1.305	6.423
35	2.108	8.004	0.005	4.980	4.290	90.730	1.326	6.560

The mean error estimated for the lifetime parameters: τ1 is ±0.07 ns, τ2 is ±0.21 ns, τ3 is ±0.001 ns, and ⟨τ⟩ is ±0.11.

Table 5

Fluorescence Decay of PIP–ctDNA Complex at Different Concentrations of SAIL

conc. (mM)	τ1a (ns)	τ2a (ns)	τ3a (ns)	α1	α2	α3	χ2	⟨τ⟩a
0	0.142	3.314		93.810	6.190		1.471	2.065
5	0.105	4.128		92.080	7.920		1.439	3.213
11	1.902	0.076	7.777	6.250	89.280	4.470	0.955	5.494
21	2.055	0.106	8.497	10.580	83.060	6.360	0.955	5.967
31	1.885	8.075	0.070	9.210	7.000	83.800	1.091	6.142
35	1.844	0.061	8.500	12.400	79.750	7.850	1.065	6.454

The mean error estimated for the lifetime parameters: τ1 is ±0.04 ns, τ2 is ±0.03 ns, τ3 is ±0.19 ns, and ⟨τ⟩ is ±0.21.

Figure 11

Difference in variation of τavg of PIP with increased concentrations of SAIL, PIP with ctDNA, and PIP–ctDNA complex with SAIL.

Time-Resolved Anisotropy

To determine the microenvironment and location of the PIP in the multicomponent environment, we utilized the time-resolved fluorescence anisotropy, which provided information about the structural dynamics of fluorophore in an organized assembly and biomolecule.[4,59] Fluorescence decay anisotropy is a very useful technique to study the molecular dynamics, rotational relaxation, and structural information of fluorophore because it directly provides the orientation dynamics of the excited fluorophore.[60] Therefore, in this work, we also utilized time-resolved fluorescence anisotropy decay to observe the rotational relaxation and dynamics of PIP in different mediums (SAIL micelle, ctDNA, and composite medium) and the location of the PIP in micellar medium. Figure showed the time-resolved fluorescence anisotropy decay of PIP in different medium. In aqueous medium (buffer), the anisotropy decay of PIP is found to be a single exponential with a lifetime component of 0.798 ns. Also, in the presence of the SAIL micelle, the anisotropy decay is fitted single-exponentially; however, the rotational relaxation decay time increases about 1.85 ns, which indicated that the environment of PIP was changed. The significantly enhanced rotational relaxation decay time of PIP shows the location of drug in a motion-constrained environment (Figure ). At higher concentrations of the ctDNA (1.25 mM), the decay pattern of PIP was noticeably different because it was fitted biexponentially with a longer and fast component. This observation revealed that the rotational relaxation of the PIP molecule gets hindered after binding with ctDNA, and this hindered rotation is eventually responsible for the residual anisotropy of PIP molecule.[46,59,61,62] As we add SAIL in the PIP–ctDNA complex, the time-resolved anisotropy decay was obtained in a single exponential function with a lifetime component of 2.30 ns. The pattern of anisotropy decay profile of PIP observes almost same as in SAIL micellar environment (Figure ), which strongly support our previous finding that is deintercalation of the PIP from ctDNA to SAIL micelle.

Figure 12

Time-resolved fluorescence anisotropy (τ) decays of PIP, PIP + ctDNA, PIP + SAIL, and PIP–ctDNA + SAIL.

Surface Tension Study

Surface tension is an appropriate technique to measure the micelle formation of amphiphilic molecules.[20] In this work, we have utilized surface tension method to measure the cmc of SAIL with PIP and PIP–ctDNA complex. The surface tension of SAIL allows us to understand its behavior under variable circumstances. The presence of cations, anions, water, drugs, and other molecules has a huge impact on the surface tension.[63,64]Figure demonstrates the plot between surface tension and log concentration of SAIL in different mediums (water, PIP, and PIP–ctDNA complex). As shown in the figure, a steep decrease in the linearity corresponds to the concentration range below the cmc where only monomers of SAIL existed at the solution surface. At higher concentrations, the solution surface becomes crowded with SAIL and any further addition of SAIL must arrange itself as micelles and no further change in surface tension will be detected.[65] The cmc of pure SAIL, SAIL in PIP, and SAIL in PIP–ctDNA complex were found to be 31.35, 25.30, and 24.45 mM, respectively (Figure ). In the presence of PIP, the cmc of the SAIL declined from 31.35 to 25.30 mM, which shows the incorporation of PIP in the SAIL micelle. The value of cmc obtained for SAIL–PIP (25.30 mM) coincides with the cmc value of SAIL–PIP–ctDNA complex (24.45 mM), which confirms that the PIP is dissociated from PIP–ctDNA complex to the SAIL micelle. This result again supported our spectroscopic result.

Figure 13

Surface tension of SAIL in water, PIP, and PIP–ctDNA complex.

Effect of SAIL on the Structure of ctDNA

To show better deintercalation processes, it is essential to know the effect of SAIL micelle on the ctDNA structure. Thus, the impact of premicellar and postmicellar concentration of SAIL on the structure of ctDNA was observed through circular dichroism (CD) spectroscopy. Figure A shows the CD spectra of pure ctDNA with two concentrations of SAIL: one is below cmc (10 mM) and the other is above cmc (35 mM). The CD spectrum of ctDNA shows two characteristic peaks attributed to the right-handed helicity: one is a positive peak at ∼275 nm and the other a negative peak at ∼245 nm.[66] As shown in Figure A, on the addition of SAIL concentration, no significant change was observed in the CD spectrum of ctDNA. This result showed that SAIL micelle does not affect the ctDNA structure, ensuring it is safe to use for deintercalation processes.

Figure 14

(A) CD spectra of ctDNA (100 μM) in the presence of different concentrations of SAIL and (B) CD spectra of ctDNA (100 μM) with PIP (50 μM) and in the presence of different concentrations of SAIL.

Further, we analyzed the CD spectrum of ctDNA with PIP and PIP–ctDNA complex with different concentrations of SAIL for deintercalation processes and confirmed the complete deintercalation of PIP from ctDNA to SAIL micelle. On the addition of PIP, the CD spectrum of ctDNA showed insignificant change at both the positive and negative bands (Figure ). This signifies that PIP does not disturb the stacking of the nitrogen bases and binds with ctDNA through groove binding. It is a known fact that both the positive and negative bands of ctDNA are unaffected in groove binding and show enormous change in the case of intercalative binding.[67] This result is also identical to other experiments. To confirm the complete deintercalation, we observed the CD spectrum of PIP–ctDNA complex in the presence of different concentrations of SAIL. Upon the addition of PIP, the positive ellipticity band of ctDNA was slightly increased; however, after the addition of SAIL in the PIP–ctDNA complex, the positive band decreased with increasing SAIL concentration (Figure B). At above cmc concentration (35 mM), the CD spectrum of the complex was overlapped with the CD spectrum of ctDNA, which indicates that PIP relocates from ctDNA to the SAIL micelle.[3] Consequently, the CD result suggested the complete deintercalation of PIP in the presence of the SAIL micelle.

Conclusions

Spontaneous interaction of ctDNA and PIP followed by the incorporation of PIP in SAIL micelles and deintercalation of PIP from ctDNA in the presence of SAIL micelles was successfully characterized by spectrophotometric and surface tension method. Absorption and emission spectroscopy suggested the involvement of minor groove binding between PIP and ctDNA complex formation. Incorporation of PIP in the SAIL micelle is confirmed by the change in spectra of both absorption and emission spectroscopy. Last, the complex phenomenon of micelle-driven deintercalation was established by the variation depicted in absorption and emission spectra in the micellar environment, illustrating the relocation of the PIP from ctDNA environment to the micellar environment. The locations of PIP in different environments were determined by KI-induced fluorescence quenching and steady-state fluorescence anisotropy, which confirm the transfer of PIP. Surface tension studies were also utilized to confirm the incorporation and deintercalation of PIP in SAIL, which showed decreased cmc of PIP–SAIL from 31.35 (pure SAIL) to 25.30 mM, and a similar cmc was also found in deintercalation processes. Time-resolved fluorescence and CD studies corroborate that PIP dissociated from ctDNA to the SAIL micelle. The present research on deintercalation process with SAIL provides imperative therapeutic significance through controlled release of drug from the target cell in cancer therapy.

Experimental Section

Calf thymus deoxyribonucleic acid (ctDNA), PIP and SAIL (1-butyl-3-methylimidazolium octyl sulfate ([C4mim] [C8SO4])), and phosphate salts (sodium monophosphate and biphosphate) were purchased from Sigma-Aldrich Corporation and used without further purification. ctDNA was prepared by dissolving in 10 mM phosphate buffer at pH 7.0 and stored in −20 °C. The intensity of the absorbance maxima was measured at 260 and 280 nm to ensure the concentration (molar extinction coefficients ε 6600 M–1 cm–1 at 260 nm) and purity of ctDNA. The A260/A280 ratios were found to be 1.8, which confirmed that ctDNA samples were free from protein. Millipore water was used during all conduct experiment. Stock solution of PIP (5 mM) was prepared in 10% ethanolic solution, and the concentration was determined by the measured absorbance at 342 nm using molar extinction coefficients (ε), 16 500 M–1 cm–1. The working solution (50 μM) was prepared by diluting the solution through experimental buffer solutions. The SAIL ([C4mim] [C8SO4]) was dissolved in water at a concentration of 400 mM (stock), and the cmc of SAIL in water at room temperature was determined at 31.35 mM via surface tension experiments, which was approximately similar to the previous reported data, that is, 34 mM.[14]

Analytik Jena Specord-210 spectrophotometer and Cary Eclipse fluorescence spectrophotometer (Varian, USA) were used for the measurement of the absorption and emission spectra, respectively. The time-correlated single-photon counting spectrometer (Horiba, Jobin Yvon, IBH Ltd, Glasgow, UK) was utilized to study the lifetime decay and dynamic anisotropy. CD measurements have been performed on a Jasco-715 spectropolarimeter, and for surface tension measurement, we utilized a high-precision delta–pi Langmuir microtensiometer (Kibron, Hebsinki, Finland) by Du-Nouy–Padday method. ITC measurements were performed on a MicroCal VP-ITC unit (MicroCal, Inc., Northampton, MA). The technical details are provided in the Supporting Information. The concentration of PIP was fixed 50 μM for all of the experiments except ITC (500 μM). For PIP–SAIL interaction, we used a constant concentration of PIP (50 μM) by gradually adding SAIL (2–35 mM) below cmc to above cmc. For PIP–ctDNA interaction, different concentrations of ctDNA (0.22–1.29 mM) titrated in PIP (50 μM) and for deintercalation processes constant PIP and ctDNA complex (1:25) was titrated with increasing SAIL concentration (2–35 mM). More instrumental details of all of the used techniques are provided in the Supporting Information.