Lipoplex-Mediated Deintercalation of Doxorubicin from Calf Thymus

Article pubs.acs.org/Langmuir

Lipoplex-Mediated Deintercalation of Doxorubicin from Calf Thymus DNA−Doxorubicin Complex Anupam Das, Chandan Adhikari, and Anjan Chakraborty* Discipline of Chemistry, Indian Institute of Technology Indore, Indore 453552, Madhya Pradesh, India S Supporting Information *

ABSTRACT: In this paper, we report the lipoplex-mediated deintercalation of anticancer drug doxorubicin (DOX) from the DOX−DNA complex under controlled experimental conditions. We used three zwitterionic liposomes, namely, 1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3phosphocholine (DMPC), and 2-oleoyl-1-palmitoyl-sn-glycero-3phosphocholine (POPC), which are widely different in their phase transition temperatures to form a lipoplex with calf thymus DNA in the presence of Ca2+ ions. The study revealed that DPPC being in sol−gel phase was more effective in releasing the drug from the DOX−DNA complex compared with liposomes that remain in liquid crystalline phase (DMPC and POPC). The higher extent of drug release in the case of DPPC liposomes was attributed to the stronger lipoplex formation with DNA as compared with that of other liposomes. Owing to the relatively smaller head group area, the DPPC liposomes in their sol−gel phase can absorb a larger number of Ca2+ ions and hence offer a strong electrostatic interaction with DNA. This interaction was confirmed by timeresolved anisotropy and circular dichroism spectroscopy. Apart from the electrostatic interaction, the possible hydrophobic interaction between the liposomes and DNA was also taken into account for the observed deintercalation. The successful uptake of drug molecules by liposomes from the drug−DNA complex in the post-release period was also confirmed using confocal laser scanning microscopy (CLSM).

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INTRODUCTION Doxorubicin (DOX) is one of the most powerful compounds of the anthracycline series and has the broadest spectrum of activity. It is considered to be a very effective antineoplastic agent and often recommended on its own or in combination with other agents for cancer therapy. It has been widely used as a potent anticancer drug for decades.1 In spite of the intense investigations, the mechanism of action of the drug still remains unclear.2 Recent studies have postulated that the antitumor activity of doxorubicin is attributed to its intercalation into the nuclear and mitochondrial DNA base pairs3,4 followed by the production of reactive oxygen species (ROS)5 and the inhibition of topoisomerase II.6 Pommier et al. reported that doxorubicin mainly inhibits type IIA human topoisomerase, both by poisoning and by catalytic inhibition.7 According to Minotti et al., the biological action of DOX originates from its binding with DNA. 8 Studies have reported that the intercalation strength of some of the anthracyclines depends upon their ability to form cleavable topoisomerase II and doxorubicin complexes.9 Most of the earlier reports consider the association of doxorubicin with DNA as an essential and important phenomenon for the biological activity of doxorubicin. In recent studies, Pérez-Arnaiz et al. have elaborately reported the different modes of binding of doxorubicin to calf thymus DNA (ctDNA).10 © 2016 American Chemical Society

Similar to the association phenomenon, slow rate of dissociation from DNA is one of the most essential criteria for a drug to be effective as a cancer therapeutic.11 Feigon and Denny et al. pointed out that slow dissociation rates from DNA and long residence times at particular binding sites correlate with greater cytotoxic potency and in vivo antitumor activity for intercalating agents.12,13 Therefore, it is necessary to study the association kinetics as well as the dissociation kinetics of the drug to evaluate its bioactivity accurately. Already a number of reports significantly dealt with the dissociation of intercalators from the DNA−intercalator complexes. T-jump relaxation for the measurement of fast kinetics14 and modified foot-printing technique to study the dissociation of drugs from specific binding sites15 are some of the experiments that were used earlier to estimate the dissociation kinetics of intercalators and DNA. Some research groups used caffeine to bring about deintercalation of the intercalators.16,17 A few groups employed micelles to act as hydrophobic sinks for the dissociation of drug molecules from the DNA−intercalator complexes.18−23 Earlier, Bhattacharya and Mandal extensively studied the lipid−DNA complexation and reported that upon binding with cationic lipid, ethidium bromide dissociates from DNA.24,25 It was Received: May 16, 2016 Published: July 27, 2016 8889

DOI: 10.1021/acs.langmuir.6b01860 Langmuir 2016, 32, 8889−8899

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atures (41, 24, and −20 °C for DPPC, DMPC, and POPC, respectively).43 To date, lipoplexes have been used as alternative nonviral gene-delivery vehicles or transfection agents in vitro and have promising potential for in vivo applications.44−46 The novelty aspect of our current study lies in the fact that lipids of the same head group but different chain lengths and phase transition temperatures (Tm) interact with DNA to different extents in the presence of a bivalent metal ion. This interaction leads to the deintercalation of the DNAbound drug. The rate of deintercalation is controlled by tuning the phase transition temperature, concentration of the phospholipids, time of incubation, and concentration of the metal ions. Earlier, lipoplexes found extensive use as alternative nonviral gene-delivery systems.44−46 Here, we have introduced that the Ca2+-mediated lipoplexes made of zwitterionic phospholipids with different phase transition temperatures and chain lengths act as efficient deintercalators. We have also demonstrated that after deintercalation, the drug molecules bind to the liposome surface. We conducted steady-state, timeresolved fluorescence measurement to monitor the deintercalation process. Time-resolved anisotropy measurement was carried out to understand the confinement of the drug molecules in the liposomes and liposome−DNA complexes. The circular dichroism (CD) study revealed the DNA− liposome interaction. Confocal imaging was conducted to unravel the fate of DOX after deintercalation from the minor groove of DNA.

observed that anionic surfactant failed to promote any destabilization of the probe−DNA complex, and hence no dissociation of the probe molecules from the probe−DNA complex takes place. The authors demonstrated that cationic surfactant is more effective in bringing the dissociation as compared with salt or small organic cations.24,25 This was attributed to the various modes of aggregation of the cationic surfactant upon dispersal in water. Recently, we have reported the deintercalation process of DOX from the DOX−DNA complex in the presence of different liposomes of different charges.26 The deintercalation process mediated by positively charged liposomes was attributed to the lipoplex formation upon interaction with DNA. This observation is in accordance with that reported by Bhattacharya and Mandal in the case of ethidium bromide.24,25 The lipoplex formation takes place because of the electrostatic force of attraction between the positively charged lipid and the negatively charged DNA.27 Recently, Aicart and co-workers have reported lipoplex formation between gemini cationic liposomes and plasmid DNA.28 Bhattacharya and Mandal have shown that plasmid DNA is a better candidate for lipoplex formation as compared with linear DNA.29 In the case of zwitterionic liposomes, bivalent cations (Ca2+/Mg2+/Mn2+/ Co2+/Fe2+) mediate lipoplex formation as reported by several groups.30−32 For phosphatidylcholine (PC) lipids, two different modes of binding of DNA and lipid molecules in the presence of calcium ions were invoked: (i) Ca2+ binds to the phosphate group of lipid molecules and induces a reorientation of the polar head groups so that the positively charged choline moieties interact with the phosphate groups of the DNA and (ii) Ca2+ bridges the phosphate groups of DNA and the adjacent PC lipids.33−36 Recently, Gurtovenko and Antipina have indicated that the electrostatic binding of DNA to lipid through Ca2+ ions affects the structural properties of DNA.33 The DNA has been found to be adsorbed electrostatically on the surface of the liposomes. Because electrostatic interaction is the key factor for lipoplex formation, it is expected that the liposomes formed by lipids of different chain lengths and hence different phase transition temperatures would display different affinities toward negatively charged DNA. Therefore, one may expect that lipoplex-mediated deintercalation would be different for liposomes of different phase transition temperatures. To explore this possibility, we have taken zwitterionic phospholipids of different chain lengths and phase transition temperatures to study the lipoplex formation with DNA and its impact on the DNA-bound anticancer drug DOX. We have chosen three different zwitterionic phospholipids, namely 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-snglycero-3-phosphocholine (DMPC), and 2-oleoyl-1-palmitoylsn-glycero-3-phosphocholine (POPC) and used Ca2+ to form lipoplexes with DNA. These phospholipids are lung surfactants and hence biocompatible.37 We opted out the use of cationic phospholipids to prepare lipoplexes because of the potential cytotoxicity of the cationic lipid−DNA complexes as delivery systems with some particular formulations.38−40 Recently, Dawson and co-workers have used an increased amount of serum and tuned biological functionality of the nanoparticles in the dispersion medium toward in vivo-relevant conditions to suppress cytotoxicity.41,42 All of the above-mentioned phospholipids were chosen keeping in mind their same zwitterionic head groups and almost nearly similar molecular weights (734.039, 677.933, and 760.08 for DPPC, DMPC, and POPC, respectively) but distinctly different phase transition temper-

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MATERIALS AND METHODS

Doxorubicin hydrochloride (DOX), calf thymus DNA (ctDNA), and lipids were purchased from Sigma-Aldrich and used without further purification. pBR322 plasmid DNA (pDNA) was purchased from SRL. The structures of DOX and the lipids are shown in Scheme 1. Phosphate buffer salts and CaCl2 were purchased from Merck. In all cases, we used Milli-Q water to prepare solutions. The stock solution of the drug was prepared by dissolving a weighed amount of DOX in a 0.01 M phosphate buffer (Na2HPO4 and NaH2PO4 containing 10 mM sodium chloride) of pH ≈ 7.4. Solutions of different concentrations of DOX were prepared by diluting the stock solution with an exact volume of buffer. ctDNA and pDNA were also dissolved in the phosphate buffer and kept under overnight stirring to solubilize the fibrous DNA. The concentration of the DNA solution was estimated by measuring the absorbance at 260 nm (OD at λ260). Both the DOX and DNA solutions were kept in the dark at 4 °C. The liposomes were prepared by the rapid ethanol injection method as reported earlier.47,48 In brief, the aqueous solution of DOX (2 μM) was taken in a round-bottom flask, and the temperature was kept above the phase transition temperature of the respective phospholipid. The phase transition temperatures (Tm) of DPPC, DMPC, and POPC are 41, 24, and −20 °C, respectively. The desired amounts of ethanolic lipid solutions were rapidly injected into the aqueous solution of DOX (2 μM) in different round-bottom flasks for different lipids (above their phase transition temperatures) and were equilibrated for 1 h. The lipid concentration of the final liposomal solution was 1 mM, and the injected ethanol was less than 0.5% (v/v) of the solution. A high concentration of DNA (5 mg/mL) was dissolved in the liposome solution in the presence of 1 mM Ca2+ (CaCl2) for the preparation of lipoplex. The mixture was kept overnight to allow the complex to form and get compact. Spectroscopic Techniques. Steady-state absorption spectra were taken in a Varian UV−vis spectrophotometer (model: Cary 100). Fluorescence titrations were performed using a Fluoromax-4p Spectrofluorometer from Horiba Jobin Yvon (model: FM-100). For DOX−DNA titration, we prepared a set of solutions of 2 μM DOX and different concentrations of DNA (from 0 to 1.00 mM) in different volumetric flasks. In a similar fashion, we prepared a set of solutions of 8890


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The quality of the fit was judged by reduced χ-square (χ2) values and the corresponding residual distribution. The acceptable fit has a χ2 near to unity. To collect the anisotropy decays, we used a motorized polarizer in the emission side. The emission intensities at parallel and perpendicular polarizations were collected alternatively until a certain peak difference between the parallel (I∥) and perpendicular (I⊥) decays was achieved. The same software was used to analyze the anisotropy data. The time-resolved anisotropy was described by the following equation

Scheme 1

r(t ) =

I (t ) − GI⊥(t ) I (t ) + 2GI⊥(t )

(3)

where r(t) is the rotational relaxation correlation function, I∥(t) and I⊥(t) are the parallel and perpendicular components of the fluorescence, and G is the correction factor. CD spectra were recorded using a Jasco J-815 spectrometer (Jasco, Tokyo, Japan). Far-ultraviolet (UV) (200−310 nm) spectra were recorded in a 0.1 cm path length cell (Hellma, Müllheim/Baden, Germany) using a step size of 0.5 nm, a bandwidth of 1 nm, and a scan rate of 20 nm/min. Each sample spectrum was obtained by averaging five scans and corrected with the solvent spectrum. For all of the CD experiments, we maintained the pH at 7.4, ionic strength (I) of 0.01 M, and temperature (T) at 25 °C. For the confocal imaging of samples, we used a confocal microscope from Olympus, model no. IX-83. A multiline Ar laser (gas laser) with an excitation of 488 nm was used. The observation mode was LSM, scan mode was XY, and scan direction was one-way. The liquid samples were dropped on glass slides and fixed with cover slips before imaging.

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RESULTS AND DISCUSSION Before conducting the deintercalation study, we checked the binding of DOX with bare liposomes using fluorescence titration. Figure 1a−c revealed the change in the fluorescence intensity upon addition of liposomes to an aqueous solution of DOX. We observed that the maximum increase in intensity took place in the case of DPPC liposomes (21 times) and the minimum in the case of POPC liposomes (8 times), whereas DMPC liposomes scored a moderate rise (16 times). This indicates that DPPC has stronger affinity toward DOX than that of other liposomes. The intensity became almost saturated at around 0.8 and completely seized at 1 mM lipid concentration. The binding constants for DOX−liposome systems as estimated following our earlier publications26 (the detailed methodology is given in the Supporting Information) were 4.70 × 108 M−1 for DPPC liposomes, 2.70 × 108 M−1 for DMPC liposomes, and 2.90 × 108 M−1 for POPC liposomes (Figure 1e). We may explain the different binding constants of DOX with liposomes in terms of the structural difference of the hydrophobic chain length and hence their pretransition temperatures. The pKa value of a single ionisable amine group of DOX is around 8.30.49 Thus, under physiological conditions (i.e., at experimental pH 7.4), DOX molecules predominate as cationic species and are bound to the head groups of liposomes through electrostatic interaction. As mentioned already, DPPC remains in sol−gel state at room temperature and DMPC and DOPC remain completely in the liquid crystalline phase. So, DPPC is least hydrated, that is, the driest among all lipids under the experimental conditions. The electrostatic binding of the liposomal head groups of DMPC and DOPC to DOX is weakened because the head groups of these two liposomes are screened by the interfacial water molecules. Moreover, the head groups of DPPC are more

liposomes where the DOX concentration was 2 μM and the liposome concentration was varied up to 1 mM. For the deintercalation process, we varied the concentration of liposomes while keeping the concentrations of DNA and DOX fixed. The samples were excited at 445 nm (λex = 445 nm), and the emission peak appeared at 595 nm (λem = 595 nm). The fluorescence spectra were corrected for the spectral sensitivity of the instrument. The excitation and emission slits were 2/2 nm for all the emission measurements. Throughout the entire titration experiments, we maintained the pH at 7.4, ionic strength (I) of 0.01 M, and temperature (T) at 25 °C. For time-resolved decays, a picosecond time-correlated single photon counting (TCSPC) machine from Horiba (Fluorocube-01NL) was used. The experimental setup for TCSPC has been described elsewhere.47,48 The samples were excited at 445 nm using a picosecond diode laser (model: Pico Brite-405L), and the decays were collected at 595 nm at magic angle (54.70°) polarization using a photomultiplier tube (TBX-07C) as the detector, which has a dark count of less than 20 cycles per second. The full width at halfmaximum (FWHM) of the instrument response function of our setup was ∼140 ps. The data were analyzed using IBH DAS version 6 decay analysis software. We maintained the pH at 7.4, ionic strength (I) of 0.01 M, and temperature (T) at 25 °C throughout the entire titration experiment. The decays were fitted with the nonexponential function n

D(t ) =

⎛ −t ⎞ ⎟ ⎝ τi ⎠

∑ ai exp⎜ i=1

(1)

where D(t) denotes the normalized fluorescence decay and ai are the normalized amplitudes of the decay components τi, respectively. The average lifetime was obtained from the equation n

⟨τ ⟩ =

∑ aiτi i=1

(2) 8891


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Figure 1. (a)−(c) Emission spectra of DOX in the presence of DPPC, DMPC, and POPC liposomes, respectively. (d) Overlapped emission spectra of DOX in the presence of three different liposomes. (e) Plot of fluorescence intensity (λex = 445 nm and λem = 590 nm) of DOX as a function of total concentration of liposome. Each curve represents the nonlinear regression fit to the experimental data using eq 6 (Supporting Information). (f) Time-resolved decay curves of DOX in the presence of different liposomes. The experiments were carried out at 25 °C, pH 7.4, 0.01 M ionic strength, and 2 μM DOX concentration.

Figure 2. (a) Time-resolved anisotropy decays of DOX at different concentrations of liposomes: (a) DPPC, (b) DMPC, and (c) POPC. (d) Comparison of time-resolved anisotropy decays of DOX at 1 mM concentration of different liposomes. The experiments were carried out at 25 °C, pH 7.4, 0.01 M ionic strength, and 2 μM DOX concentration.

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Figure 3. Deintercalation of DOX from the DOX−DNA complex at different liposome concentrations in the lipoplexes. (a) DPPC (in the inset, the quenching spectra of the DOX fluorescence intensity due to DNA intercalation are shown), (b) DMPC, and (c) POPC. The arrows indicate the enhancement of DOX fluorescence intensity upon increasing the concentration of liposome in the lipoplex. (d) Overlapping spectra of DOX at 1 mM concentration of different liposomes in different lipoplexes. All the deintercalation experiments were carried out at 25 °C, pH 7.4, 0.01 M ionic strength, and 2 μM DOX concentration.

extended toward water owing to their smaller size and hence capture a larger number of DOX molecules. Being in the liquid crystal phase, the head group regions of DMPC and DOPC have a higher fluctuation than that of DPPC. The larger fluctuation in the head group region may be a factor for the weak interaction of DOX with the DMPC and DOPC liposomes. The stronger binding of DOX to the DPPC liposomes is further supported by the lifetime data of DOX in the presence of different liposomes of different chain lengths. The representative decays of DOX in the presence of different concentrations of liposomes are shown in Figure 1f. We observed a biexponential lifetime decay of DOX in all of the liposomes. This observation is consistent with our recent report.26 The complex lifetime decay is primarily attributed to the heterogeneous distribution of the DOX molecules in the liposomes. The time components are composed of a shorter component (τ1) around 1.1 ns and a longer component (τ2) around 4.1 (76%), 3.55 (68%), and 3.00 (66%) ns for DPPC, DMPC, and POPC liposomes, respectively (Table S1a−c). We ascribe the longer component as liposome-bound DOX. Notably, the time constant and amplitude of the liposomesbound DOX are the highest in DPPC and the lowest in POPC liposomes. The order of the lifetime is in accordance with the phase transition temperature of lipids. This observation led to the same conclusion drawn from the steady-state measurement that the binding of DOX with liposomes of a higher phase transition is more facile than that with liposomes of a lower phase transition temperature. The lifetime decay measurement was followed by time-resolved anisotropy measurement, which also leads to a similar conclusion. The representative anisotropy

decays are shown in Figure 2. It is revealed that the rotational relaxation time increased upon increasing the concentration of liposome for all three liposomes. The order of increments is DPPC > DMPC > POPC liposomes. Because DPPC remains in the sol−gel phase at room temperature, the electrostatic interaction would be the highest for DPPC. Moreover, the DOX molecules have more confinement in the rigid vicinity of the DPPC liposomes owing to its sol−gel character at room temperature. Because there is no residual anisotropy, it may be concluded that the global motion of any of the liposomes is not coupled with the rotation of the DOX molecules. Having sufficient information regarding the binding of DOX with different liposomes, we move on to our main objective, which is deintercalation of DOX from the DNA−DOX complex by different liposomes. We previously reported that in the presence of DNA, almost 95% of quenching takes place in the fluorescence intensity of DOX.26 The extreme amount of the quenching of the DOX intensity in the presence of ctDNA is attributed to the formation of nonfluorescent complexes (PD1 and PD2) through the intercalation of aromatic DOX core to the guanine-cytosine-rich region of a DNA double helix. In the deintercalation process, the drug molecules come out of DNA in the presence of liposomes. So, during the deintercalation study, we first varied the concentration of liposomes to understand the mechanistic pathway of the deintercalation process using steady-state and time-resolved measurements. Please note that the deintercalation study was conducted in the presence of Ca2+ ions for the formation of lipoplexes. In the second step, we monitored the controlled release of drug molecules from the DNA−DOX complexes for 8893


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Figure 4. (a) Time-resolved decays of DOX−DNA upon varying the concentration of liposomes in different lipoplexes. (b) Average lifetime ⟨τ⟩ with increase in the concentration of liposomes in different lipoplexes. The experiments were carried out at 25 °C, pH 7.4, 0.01 M ionic strength, and 2 μM DOX concentration.

Figure 5. (a) DOX release profile from the DOX−ctDNA complex in the case of different lipoplexes. (b) DOX release profile from the DOX− pDNA complex in the case of different lipoplexes. (c) Comparison between DOX release profiles from the DOX−ctDNA complex in the case of DPPC lipoplex (in the presence of Ca2+) and bare DPPC liposome (in the absence of Ca2+). (d) Comparison between DOX release profiles from the DOX−ctDNA complex in the case of DMPC lipoplex (in the presence of Ca2+) and bare DMPC liposome (in the absence of Ca2+). The timedependent experiments were carried out at 25 °C, pH 7.4, 0.01 M ionic strength, and 2 μM DOX concentrations.

fluorescence intensity of the DOX−DNA solution increases upon addition of liposomes, which means that the drug molecules come out from the minor groove of the DNA. We note that the maximum amount of drug release was recorded in the case of the DPPC lipoplex (215% rise in intensity), while the minimum (76% rise in intensity) was recorded in the case of the POPC lipoplex, and DMPC scored a moderate drug release (138% rise in intensity). Time-resolved measurements were also used to monitor the changes in lifetime upon the deintercalation process. Timeresolved studies reveal changes similar to those in steady-state

different lipoplexes with respect to time. Here, pDNA and ctDNA have been used to see the effect of added liposomes on the DNA-bound drug. We conducted time-resolved anisotropy and CD study to unravel the liposome−DNA interaction. Finally, we conducted the lipoplex-mediated deintercalation study using confocal imaging, which unambiguously reveals the time-dependent deintercalation process and post-release fate of DOX. Let us start the discussion with the results obtained from the steady-state measurement. Figure 3 depicts the effect of liposomes on the DOX−DNA solution. We observe that the 8894


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Figure 6. Time-resolved anisotropy decays of DOX of DNA−DOX complex at different concentrations of liposomes for different lipoplexes. (a) DPPC lipoplex, (b) DMPC lipoplex, and (c) POPC lipoplex. (d) Comparison of time-resolved anisotropy decays of DOX in different lipoplexes at a maximum concentration of liposomes (1 mM). The experiments were carried out at 25 °C, pH 7.4, 0.01 M ionic strength, and 2 μM DOX concentration.

for lipoplex formation. Therefore, it is very likely that the positively charged liposomes interact with negatively charged DNA to form the lipoplex, which is responsible for the deintercalation observed.30,50,51 The interpretation can further be extended by the argument of Bhattacharya and Mandal24,25 in the case of ethidium bromide. They demonstrated that the electrostatic binding between the liposomes and negatively charged DNA is facilitated by the positive charge on different lipids, resulting in the neutralization of the DNA backbone.24,25 This may reduce the intra- and interstrand electrostatic repulsion present in the native DNA phosphate backbone.24 As a result, DNA duplexes pack in a more compact fashion, leaving insufficient space for the accommodation of the incoming guest molecules (intercalator) or of the one preexisting within the double strand.24 This destabilizes the DNA− drug complex and results in the release of the drug. This argument justifies the higher deintercalation in the case of lipoplex formation. Now we need to address why DPPC lipoplex causes the highest deintercalation and POPC lipoplex brings in the least deintercalation. It is well-known that unsaturated lipids such as POPC display a much lower affinity toward bivalent cations such as Ca2+.56−65 Thus, only a small fraction of the unsaturated lipids absorb bivalent cations and become positively charged. On the other hand, liposomes composed of saturated lipids display a larger affinity toward bivalent cations.56,66 This affinity further increases because of the smaller head group area of the saturated lipids. The smaller area per PC head group results in more-extended conformation of the head groups toward water. Therefore, the interaction of DNA with the POPC liposomes is weaker than the interaction

measurement and confirm that liposome brings about the deintercalation of DOX from the DNA−DOX complex. It is observed that the average lifetime of DOX increases upon increasing the liposome concentration because of the formation of lipoplex. The increments in ⟨τ⟩ of DOX for different lipoplexes (DPPC, DMPC, and POPC) are 86, 57, and 40%, respectively (Figure 4 and Table S2a−c). Notably, we observe that the lifetime component (τ2 = 4.32 ns) is the longest in the DPPC liposomes (τ2 = 4.32 ns) but the least in the POPC liposomes (3.17). The DMPC liposome scores a moderate value (3.66 ns). The increment in τ2 and its amplitude values takes place because of the postrelease interaction of DOX with the liposome. Finally, we conducted a time-dependent release study for both ctDNA and pDNA with the help of steady-state fluorescence measurement. Figure 5 illustrates that drug release takes place over a maximum time period of 100 min. All of the above observations confirm that the deintercalation of DOX occurs from DNA upon lipoplex formation. Interestingly, for both ctDNA and pDNA, the extent of deintercalation is different for different lipoplexes (Figure 5a,b). Whereas the highest percentage of drug release is caused by the DPPC− DNA lipoplex, the lowest one is displayed by the POPC−DNA lipoplex (Figure 5a,b). Interestingly, Figure 5c,d reveals that the extent of drug release by bare liposomes is much less when compared with that of their respective lipoplexes. The binding of Ca2+ to lipids occurs via phosphate moiety on the head group region of the lipid.30,50−55 Thus, in the presence of Ca2+ ions, the liposome becomes positive in nature. One Ca2+ ion bridges two adjacent phosphate groups: one from the phospholipid head group and another from the DNA backbone 8895


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Figure 7. (a) CD spectra of ctDNA in the case of different lipoplexes. (b) Comparison of the CD spectra of ctDNA in the case of DMPC lipoplex (in the presence of Ca2+) and bare DMPC liposomes (in the absence of Ca2+). (c) Comparison of CD spectra of ctDNA in the case of DPPC lipoplex (in the presence of Ca2+) and bare DPPC liposomes (in the absence of Ca2+). The CD experiments were carried out at 25 °C, pH 7.4, and 0.01 M ionic strength.

relaxation time takes place in the DPPC−DNA complex while the minimum is observed in the POPC−DNA complex. The slowest rotational relaxation in the DPPC−DNA complex originates from the most compact and rigid lipoplex formation with DNA owing to the dryness of DPPC in the head group region and gel phase at the experimental temperature. The rigidity of the lipoplex is least in the case of POPC because POPC remains in a perfect liquid crystalline phase, which weakens the rigidity of the complex, resulting in faster rotational relaxation. The stronger interaction of the DPPC liposome with DNA is further substantiated by the CD results. Figure 7 shows the CD spectra of the DNA−DOX complex upon interaction with different liposomes. The DNA−DOX complex exhibits two positive peaks at 220 and 279 nm and a negative peak at 246 nm in the CD spectra. Maximum deviation (or change) in the CD spectra was observed in the case of DPPC lipoplex whereas minimum deviation was observed in the case of POPC lipoplex. The intermediate spectra correspond to DMPC. More the deviation in the spectra of DNA−DOX, higher the distortion and deformation of DNA−DOX helix. We concluded from the CD spectra that maximum distortion of DNA−DOX takes place in the case of DPPC lipoplex and the least is observed in the case of POPC lipoplex. To have a better insight into the DNA− liposome interaction, we conducted CD measurement for a particular DNA−liposome system in the presence and absence of Ca2+ ions. Figure 7b,c depicts that the interaction of DNA with the DMPC and DPPC liposomes is stronger in the presence of Ca2+ ions, which is obviously due to the lipoplex formation. Adsorption of Ca2+ ions facilitates the lipid chain packing by increasing the transition temperature (for DPPC and DMPC, the phase transition temperatures increase from 23 to 29 °C and 41 to 48 °C, respectively), thus stabilizing the gel phase.56−65 Therefore, the gel phase has a stronger interaction with DNA. This observation also answers why we observe a maximum interaction with DPPC among all the liposomes. We further carried out zeta potential measurement experiments by titrating the DOX−ctDNA complex at different concentrations of Ca2+ for the three lipoplexes (Supporting Information Figure 1). It was observed that the zeta potential value increases from that of the DOX−ctDNA complex upon titration and reaches the maximum for the DPPC−DNA lipoplex. In this context, we need to discuss the role of the hydrophobic contribution due to the alkyl chain length. Bloomfield and co-workers reported that the enthalpy change

with the DMPC and DPPC liposomes because POPC displays a lower affinity towards Ca2+ ions. This results in the lowest amount of drug release by the POPC liposomes. Under the experimental conditions, DPPC remains in the gel phase (Tm of DPPC 42 °C), whereas POPC remains in the liquid crystalline phase (Tm of POPC −2 °C). DMPC remains in a nearly liquid crystalline phase because its Tm is 24 °C. Thus, DPPC is the least hydrated among all of the lipids used under the present experimental conditions. The gel-phase membranes bind more divalent cations than do the liquiddisordered membranes (melted acyl chains). For DPPC in the gel phase, the area per lipid head group is around 47 Å2, whereas DMPC, being in the liquid crystalline phase, has a lipid area of around 64 Å2.67,68 Therefore, more head groups of DPPC are exposed to water, which captures a larger number of cations. Therefore, DPPC liposomes offer stronger interaction toward DNA than do the head groups of DMPC. Again, the rigid DPPC head groups, upon formation of the lipoplex, deform or distort the DNA double-helix stability to a greater extent when compared with other lipids and destabilize the drug−DNA complex. Both the DMPC and POPC liposomes are “softer” than the DPPC liposome. “Softer” liposomes are less prone to deform or disturb the stability of DNA double helix. All of these factors together are responsible for the observed higher deintercalation of DOX from the DOX−DNA complex caused by the DPPC liposome. The driving force for the lipoplex formation is purely due to the electrostatic interaction between the DNA and liposomes. The electrostatic interaction is the strongest in the case of DPPC liposome because it is the driest liposome among all three and hence its head groups are not screened by water molecules. It has been reported that adsorption of DNA on the lipid surface leads to increased ordering of lipid tails. Dawson and co-workers reported that upon lipoplex formation, the main transition temperature of the bound lipid becomes higher, which implies a more-ordered phase.34 In the present case, DNA−DMPC and DNA−POPC interactions are weaker than the DNA−DPPC system because of the increased fluctuation of the head groups in the disordered bilayer. To validate this assumption, we conducted time-resolved fluorescence anisotropy and CD studies, which unambiguously reveal that DPPC has a stronger interaction with DNA when compared with the other two liposomes. Figure 6 reveals that the rotational relaxation of DOX becomes slower in all of the lipoplexes than that in the DOX−DNA system. The maximum increment in rotational 8896


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Langmuir upon binding of surfactant with DNA remains the same with the alkyl chain length, and this endothermic enthalpy was found to oppose the DNA−lipid binding.69 However, the entropy due to hydrophobic interaction is positive and increases with the chain length of the surfactant. This hydrophobic contribution of entropy is the governing factor for the overall Gibb’s free energy change. In the present case, DPPC has a chain length longer than that of DMPC.69 Thus, it is expected that the hydrophobic contribution of DPPC to the overall free energy change would be higher than those of DMPC and POPC. The very high binding affinity of DOX to the liposomes could be because the liposomes act as the “hydrophobic sink” for the drug-like micelles. Probably, liposome−DOX binding gets the entropic advantage as mentioned in the case of SDS micelles.26 This conjecture is in accordance with the observation that the DPPC lipoplex brings about higher deintercalation when compared with other liposomes. We earlier reported that the addition of alcohol to the DOX−DNA solution brings about the deintercalation process because alcohol changes the hydrophobicity and polarity of the system.26 Alcohol reduces the dye affinity for DNA and lowers the association affinity of the intercalator with DNA.70 This fact also indicates that DPPC and DMPC are more hydrophobic when compared with POPC because of their higher phase transition temperatures and difference in their prehydration levels. The ET(30) values obtained for DPPC, DMPC, and DOPC liposomes are 41.7, 47, and 54 kcal/mol, respectively.71 From this, we conclude that DPPC induces more nonpolarity effect in the solution and causes more deintercalation as compared with DMPC and DOPC. Time-dependent DOX release and fate of DOX in the postdeintercalation phase were further confirmed with the help of confocal imaging of DOX-encapsulated lipoplexes at different time intervals (Figure 8). Figure 8a represents the control experiment image. Figure 8b represents the image corresponding to zero time, that is, immediately after the addition of liposomes to the DOX−DNA complex. At zero time, it is observed that DOX is not bound to liposomes because there is no signal emitting from the lipoplex. As time elapses, DOX molecules come out of the DNA−DOX complex and bind to the liposomes (Figure 8c,d). This observation unambiguously confirms the deintercalation of drug molecules from DNA and the uptake of drug molecules by liposomes in the post-release period.

Figure 8. Confocal microscopy images (a) Only liposome as control (without DOX encapsulation) giving no signals, (b) DPPC liposomes at zero time (no DOX being encapsulated) giving no signals, (c) image taken at 30 min of incubation. Accumulation of DOX by liposome initiates. DOX-accumulated liposomes give a very low intensity signal. (d) Image taken at infinite time incubation in DPPC lipoplex. Liposomes accumulate almost all the DOX from the DOX−DNA complex and give the maximum intensity signal.

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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.langmuir.6b01860. Time-resolved data for DOX upon varying concentrations of DPPC, DMPC, and POPC liposomes, timeresolved data for the three lipoplexes upon increasing the concentrations of the corresponding liposomes, zeta titration curves for different lipoplexes upon increasing the concentrations of Ca2+ (PDF)

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AUTHOR INFORMATION

Corresponding Author

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*E-mail: [email protected]. Phone: +91-731-2438773.

CONCLUSIONS In conclusion, we demonstrated that the lipoplex formation results in the deintercalation of drug molecules from the drug− DNA complex. We found that the degree of deintercalation depends on the electrostatic interaction between DNA and the lipid head groups. The saturated liposomes appeared to be better candidates for the deintercalation process than the unsaturated complex. Further, the liposomes in the sol−gel phase, because of their smaller head groups, exhibit higher affinity toward DNA, resulting in a higher extent of deintercalation when compared with liposomes in the liquid crystalline phase. We also found that the extent of deintercalation is less in the absence of Ca2+ ions, suggesting that deintercalation is facilitated by the formation of lipoplex. The DOX molecules bind to the liposomes in the postdeintercation phase.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank SIC, IIT Indore, for providing the facility and infrastructure. A.D. and C.A. thank IIT Indore for providing fellowship.

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REFERENCES

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