Mitoxantrone (1,4-dihydroxy-5,8-bis[2-(2-hydroxyethylamino)ethylamino]anthracene-9,10-dione) is a synthetic anthracenedione anticancer drug developed in the 1980s as a doxorubicin analogue in a program to find drugs with improved antitumor activity and decreased cardiotoxicity compared with the anthracyclines [1
]. It is the only drug of the anthracenedione class approved for clinical use. Mitoxantrone is used primarily in therapy for breast cancer, acute leukemia, lymphoma and prostate cancer and, more recently, in the active forms of relapsing-remitting or secondary progressive multiple sclerosis [2
]. Previous studies suggest that mitoxantrone has less cardiotoxicity than anthracyclines at equivalent doses, but further investigations revealed that mitoxantrone presents a similar cardiac toxicity clinical profile to doxorubicin and that the toxicity can occur at any time during therapy, and the risk increases with increased cumulative dose [5
The mechanism of the mitoxantrone-associated cardiotoxicity is still poorly understood and can involve formation of reactive oxygen species [8
], altered function of myocardial adrenergic receptors [11
], multiple disturbances in calcium homeostasis [12
], impaired expression of various important cardiac proteins [13
], proteome changes including profound impairment of mitochondrial energy production, perturbations in energy channeling and impairments of mitochondrial antioxidant protection [14
The antitumor activity of mitoxantrone is related to its ability to bind to DNA and to inhibit both DNA replication and DNA-dependent RNA synthesis [15
]. Besides, mitoxantrone is also a potent inhibitor of topoisomerase II, which is an important enzyme for the repair of damaged DNA and this results in single and double strand breaks [3
]. Structurally mitoxantrone is symmetrical, containing a tricyclic planar chromophore substituted with two nitrogen-containing side chains (Figure 1
). The planar anthraquinone ring is the key element for mitoxantrone molecule intercalation between the base pairs of DNA, whereas the basic side groups contribute to the electrostatic binding with the negatively charged DNA phosphate backbone [23
The drug molecule has to pass through the cellular and nuclear membranes and to interact with them before approaching the target DNA inside of the cell. The exact manner in which bioactive molecules can interact and penetrate cellular membranes, and molecular details of these mechanisms are foremost to their chemotherapeutic action because the membrane acts as a barrier to the permeation of polar molecules and this effect is mainly due to the hydrophobicity of the membrane interior [24
]. It is therefore interesting to see how different membrane parameters like surface charges, hydrophobic effect, and length of the fatty acid chain would affect the structure of mitoxantrone and its interaction with membranes.
Biological membranes are extremely complicated dynamic structures due to the existence of lipid domains, lipid asymmetry, coexistence of phases and diversity in lipid composition. Also, the complexity of biological membranes is further increased by their association with proteins and carbohydrates. Because of this complexity, the interactions between biological membranes and bioactive ligands are very difficult to investigate in a real situation [25
]. Therefore, simplified model membranes in which the organization best mimics the bilayer lipid arrangement found in natural membranes have been developed [26
Micelles with their hydrophilic surface and hydrophobic interior serve as simple membrane mimetic systems that allows controlled studies of the effect of different membrane parameters on the binding of drug molecules [28
]. The use of surfactant micelles as membrane models is considered to be more advantageous, compared to liposomes and soluble polymers, because of their simplicity, low toxicity, tunable charge, narrow size distribution, longer residence time in the system and the enhanced bioavailability and stability of drug through micelle encapsulation [29
]. Besides being used as biomembrane model systems, micelles can be used to solubilize poorly soluble drugs, thus increasing their bioavailability. Micelles are known to have an anisotropic water distribution within their structure; the water concentration decreases from the surface towards the core of the micelle, with a completely hydrophobic (water-excluded) core [31
]. Consequently, the position of a solubilized drug in micelles depends on its polarity: nonpolar molecules will be solubilized in the micellar core, and drug molecules with intermediate polarity will be distributed along the surfactant molecules in certain intermediate positions [28
Ionic (sodium dodecyl sulfate (SDS), cetyltrimetylammonium bromide (CTAB)) and non-ionic surfactants (Triton X-100, Brij-35 and Tweens) are commonly accepted as model systems for studying different aspects of membrane interactions with drug molecules, including their localization [33
]. Because many biological processes occur at the ionizable surface of membranes or in the hydrophobic region, a comparative study of the drug interaction with different charged surfactants may provide information about the nature of the binding forces involved in the drug-membrane interaction [32
The change of the pharmacological behavior of drugs by their encapsulation in micelles can be a way to improve the treatment efficacy and to overcome the toxic side effects of drugs, ensuring the transport to specific sites of action without loss. Systematic studies in this field must involve the evaluation of drug interaction with biological membranes. The main reason for this is that the nature and magnitude of drug/biomembrane interaction can determine the drug release from the carrier [36
]. Different drug-delivery systems have been studied in an attempt to improve the antitumour effect of mitoxantrone and to prevent its harmful side effects [37
As the surfactant micelles can be used as drug carriers and simplified model membranes in order to obtain information about the interaction with biological membranes, a systematic investigation of the interaction of mitoxantrone with anionic (SDS) [41
], cationic (CTAB) [42
] and non-ionic (Triton X-100, Tween-20, Tween-80) [43
] surfactants has been performed. Ionic micelles were chosen as a model of the lipid bilayer in order to investigate the electrostatic contribution to the drug binding (the influence of different charges at the polar surfactant head groups), while the non-ionic micelles were chosen in order to evaluate the hydrophobic contribution to mitoxantrone binding.
This review provides an overview on these studies regarding the interaction of mitoxantrone with different surfactants. UV-Vis absorption spectroscopy has been used to evaluate the parameters such as binding constants, partition coefficients, free energy of interaction and also to predict the location of drug molecules in the micelles. Quantitative understanding of the drug-micelle interactions is an important step in the design of efficient drug delivery systems.
2. UV-Vis Absorption Studies
The surface of biological membranes frequently presents a net charge due to the ionisable head groups of lipids. Therefore, the binding characteristics of charged and uncharged drug molecules may be very different [44
]. Surfactant micelles bearing different charges on their surface can be used in UV-Vis absorption spectroscopy studies in order to obtain qualitative and quantitative information about drug-micelle interactions.
Mitoxantrone is a weakly basic drug with two ionizable amine groups (pKa values of 8.3–8.6) [45
] and its distribution will be influenced by the micro-environmental pH. pKa shifts were observed for different drugs upon their binding to micelles or bilayers [32
]. The pH-dependent behaviour of the mitoxantrone-surfactant interaction was evaluated in vitro at pH 7.4, when mitoxantrone is a dication with two positive charges on the nitrogen atoms from the side chains [19
] and pH 10, when it is uncharged due to the deprotonation of the side chain amino groups [48
The visible absorption spectrum of mitoxantrone in phosphate buffer pH 7.4 consists of three overlapping spectral absorption bands: two absorption bands at 660 and 610 nm, and a shoulder at about 570 nm, more evident at higher drug concentrations. The shape of the absorption spectrum of mitoxantrone is dependent on concentration and, as this dependence is usually assigned to the formation of molecular aggregates, the band at 660 nm was assigned to the monomer (M), the band at 610 nm to the dimer (D) and the band around 560 nm to the formation of the higher aggregates of the drug [49
]. In carbonate buffer pH 10, the absorption maxima of dimer and monomer are red shifted and the ratio of monomer to dimer absorbances decreases, indicating that the dimerization process is favored in a basic environment (Table 1
]. Also, at both pH values, the positions of the absorption maxima of dimer and monomer do not change with temperature, but the ratio of monomer to dimer absorbance increases with temperature, indicating the dissociation of mitoxantrone aggregates (dimers or higher aggregates) with increasing temperatures [50
The absorption spectral behavior of mitoxantrone shows a strong dependence on surfactant type and concentration. In the case of SDS [41
], the intensity of both the monomer and dimer absorption bands decreases for surfactant concentrations lower than the critical micellar concentration (CMC). When the SDS concentration is lower than the CMC, the cationic mitoxantrone molecules are strongly attracted by the anionic SDS molecules and the decrease of absorbance was assigned to the neutralization of mitoxantrone charges by electrostatic interaction between the positively charged amino groups of the drug and the negatively charged surfactant groups, allowing the formation of a drug–SDS ion-association complex. The electrochemical results [41
] and Job’s method of continuous variation and molar ratio method [51
] indicated that the stoichiometry of the mitoxantrone-SDS complex is 1:2. Due to the neutralization of the charges of the mitoxantrone dication in the ion-association complex, the mitoxantrone molecule becomes more hydrophobic and its dimerization increases [41
Unlike the anionic surfactant SDS, the presence of premicellar CTAB and non-ionic surfactants concentrations do not change the absorption spectrum of mitoxantrone [42
]. For surfactant concentrations higher than the CMC, the intensity of both monomer and dimer bands increases, but the monomer absorbance at 660 nm becomes predominant. Also, for micellar surfactant concentrations both absorbance maxima are red shifted (Table 1
). This bathochromic shift indicates the interaction between mitoxantrone and surfactant micelles and the transfer of mitoxantrone molecules are from the highly polar aqueous phase into a relatively nonpolar micellar environment. The increase in the absorption maxima at 660 nm with the increase of surfactant concentration above CMC is due to the interaction of mitoxantrone with micelles and this interaction induces the dissociation of the mitoxantrone aggregates. Thus, mitoxantrone molecules are encapsulated in micelles as monomer, following the equilibria: mitoxantrone aggregates ↔ mitoxantrone monomer (bulk) ↔ mitoxantrone monomer (micelles), similar to the pinacyanol cationic dye [52
]. Therefore, the presence of micelles causes a shift this equilibrium to the right and the conversion of mitoxantrone aggregates to monomer and encapsulation of mitoxantrone monomers in micelles.
Theoretical calculations were carried out with the Gaussian 03 software package [54
] using the B3LYP/6-311G* basis set in order to predict the molecular structure of mitoxantrone monomer and dimer. Water solvent effects were simulated by the conductor-like polarizable continuum model (CPCM) [55
]. Optimized geometries of monomer mitoxantrone in neutral and dication form are presented in Figure 2
. When the mitoxantrone monomer has two positive charges on the side chain nitrogen atoms (like in the experimental conditions at pH 7.4), the molecule has both side chains almost in the same plane as the aromatic rings (the dihedral angle between them being 178 degrees) and the highest distance between two side chains is 2.03 nm. When mitoxantrone monomer is not charged (like in experimental conditions at pH 10), the molecule does not have a fully planar structure, the amino alkyl side chains are entirely out of plane, with a 81 degrees out of planarity of the whole geometry, close to the 77 degrees from literature data [56
]. The two side chains are closer to each other, the distance between them being 1.35 nm.
In the mitoxantrone dimer geometrical arrangement (Figure 3
), the planes of the drug chromophores are parallel to each other due to π-π stacking interactions and situated at 0.47 nm apart and this value is close to the distance between two pyrene molecules (0.42 nm) [57
]. This geometric arrangement of mitoxantrone dimer in solution resembles the calculated structures proposed using NMR analysis [58
Because a typical micelle has a diameter about 5 nm [59
] and due to the antiparallel orientation of alkyl side chains in the dimer geometry, it is difficult for mitoxantrone dimer to be entrapped in micelles, thus we assumed that mitoxantrone is encapsulated in micelles in monomer form.
The CMC values for all surfactants in the presence of mitoxantrone were determined from the change in the absorption spectrum of mitoxantrone and are smaller than the values in pure water, knowing that the CMC is influenced by the presence of different ions and molecules [60
when mitoxantrone monomers are encapsulated in micelles [42
]. The variation of absorbance at 660 nm as a function of SDS and CTAB concentration at pH 7.4 and 10 is presented in Figure 4
. In the case of SDS two distinct processes depending on the surfactant concentration are observed at pH 7.4: process I in premicellar range, assigned to the electrostatic interaction between positively charged mitoxantrone molecules and negatively charged SDS monomers and process II in micellar surfactant concentrations, when the SDS micelles are formed and the drug is encapsulated in micelles in monomer form [41
]. For CTAB, Triton X-100, Tween-20 and Tween-80 surfactants only one process is observed for micellar surfactant concentration
Erdinc et al. studied the interaction of the cationic anthracycline drug epirubicin with anionic (SDS), cationic (CTAB) and non-ionic (Triton X-100, Tween-20) surfactants by using UV-Vis absorption spectroscopy [61
]. Their results indicated that in the case of SDS, epirubicin-SDS molecular complex formation takes place at premicellar surfactant concentrations due to electrostatic interactions followed by dissociation and further incorporation at micellar concentrations. Also, it was found that the binding constant is higher for non-ionic surfactants than SDS and a very week interaction occurred between epirubicin and CTAB due to electrostatic repulsion [61
Zafar and co-workers reported the interaction of some anticancer uracil derivatives with SDS and CTAB surfactants [62
]. Cyclic voltammetry and UV-Vis spectroscopic techniques were used to evaluate the binding constants, partition coefficients between bulk and micellar phase, and the number of drug molecules incorporated per micelle. These studies revealed that at premicellar concentrations, the binding is mainly due to the electrostatic interactions between the surfactant monomers and the drug molecules, while in the postmicellar region, drug is encapsulated into micelles due to electrostatic as well as hydrophobic interactions [62
Studies performed by Bhattacharjee et al. [63
] indicated that the ionic mixed micelles of Tween-80-NaDC (sodium deoxycholate) can encapsulate the cationic drug doxorubicin by noncovalent electrostatic interaction and doxorubicin encapsulated in these mixed micelles has greater anticancer activity in different cancer cell lines as compared to doxorubicin solution. Also, the electrostatic binding between doxorubicin and anionic surfactant aerosol OT leads to the formation of a hydrophobic drug-surfactant complex and this complex can be encapsulated in Pluronic block copolymer (P123) micelles without disrupting the structure of aggregates [64
Cyclic voltammetry investigation of the interaction between doxorubicin and SDS outlined a weak predominantly electrostatic drug-surfactant interaction in the range of pre-micellar and micellar concentrations of surfactant [65
Spectral studies regarding the interaction of 2-amino-3-hydroxyanthraquinone (AQ), an analogue of the anthracycline anticancer drugs, with SDS and CTAB showed that the hydrophobic interactions play a crucial role in the binding of AQ to SDS micelles, while the hydrophilic interactions plays an important role in its interaction with CTAB micelles [66
Spectral and electrochemical investigation of the interaction of anticancer drug actinomycin D (actinomycin D contains a 2-aminophenoxazin-3-one chromophore and two cyclic pentapetide lactones) with different surfactants indicated significant differences in the strength of the interaction, the binding constants being higher for charged surfactants (SDS, CTAB) than non-ionic surfactants (Triton X-100) [67
]. Also, the binding constants for the interaction of actinomycin D with CTAB, SDS and Triton X-100 are much smaller than the binding constants for the interaction of mitoxantrone with these surfactants.
In order to quantify the evolution of the three overlapping absorption components of mitoxantrone in different conditions, the deconvolution of the spectra in elementary bands was performed using the Gaussian multi-peaks function in the PeakFit 4.11 software. The goodness of the fit was considered from the fitting parameter (R2
~1) and the symmetrical distribution of the residuals. Figure 5
shows the variation of monomer, dimer and higher aggregate components of mitoxantrone in phosphate buffer pH 7.4, carbonate buffer pH 10 and in the presence of micellar concentrations of SDS (8.64 × 10−3
M), CTAB (3.88 × 10−3
M), Tween-20 (3.12 × 10−2
M), Tween-80 (3.46 × 10−2
M) and Triton X-100 (2.68 × 10−2
M), obtained from deconvolution of the spectra.
It can be observed that for both pH values, the dimer component percent is almost constant (about 45%), but at pH 10 the higher aggregates component increases on expense of monomers, indicating that the aggregation of mitoxantrone monomers is favored in basic medium and dimers and higher aggregates are the predominant species. For pH 7.4 and 10 and micellar concentrations of SDS, CTAB, Tween-20, Tween-80 and Triton X-100, the monomer and dimer components increases on expense of higher aggregate component, indicating the dissociation of these species caused by the interaction of mitoxantrone with surfactant micelles (monomer ↔ dimer ↔ higher aggregate equilibrium is shifted to the monomer and dimer formation). It can also be observed that for both pH values, the presence of anionic, cationic and non-ionic surfactant micelles induces the disaggregation of mitoxantrone and the percent of monomer, dimer and higher aggregate components is almost the same for all investigated surfactants.
The interaction of mitoxantrone with surfactant micelles induces the dissociation of drug aggregates similar with the interaction of mitoxantrone with DNA [69
]. This similar behavior can be explained by analogy between DNA and SDS micelles (negatively charged surface and hydrophobic interior) as receptor for intercalating drugs. Digman and co-workers investigated the interaction of intercalating drug daunomycin with surfactant micelles as a model for the hydrophobic contribution to the free energy of DNA intercalation reactions [70
Absorption and circular dichroism spectroscopy, and thermal denaturation studies have shown that the presence of SDS micelles induces the exclusion of intercalated mitoxantrone monomer from DNA and further their encapsulation in micelles [69
]. This deintercalation process of intercalated drug molecules from DNA in the presence of surfactant micelles and their transfer inside of micelles was observed for different ligands [71
]. Considering that amphiphilic molecules are present in real biological systems (i.e., phospholipids in the cell membranes, polyamines in nucleus, bile salts in the bile), this deintercalation process induced by micelles may have significant importance in biological processes [71
3. Polarity of the Micellar Environment and Probable Location of Mitoxantrone
A micellar structure is characterized by different layers: (a) the hydrophobic core containing the hydrocarbon tails of the surfactant molecules; (b) Stern layer containing compact head groups of ionic surfactants or the palisade layer composed of the polyoxyethylene chains of non-ionic surfactants; and (c) the surface of micelles [31
]. A drug molecule can interact with micelles in different ways, depending on the drug hydrophobicity: hydrophilic drugs can be adsorbed on the surface of the micelle, hydrophobic molecules can be trapped in the hydrophobic core of the micelles or, in the case of drugs with intermediate solubility should be located in intermediate positions within the micelle such as between the hydrophilic head groups of non-ionic micelles and in the palisade layer between the hydrophilic groups and the first few carbon atoms of the hydrophobic groups [31
]. The location of molecules into micelles determines the extent of solubilization, the pharmacokinetics and biodistribution of incorporated drug molecules [77
As seen in Table 1
, the presence of micellar concentrations of SDS, CTAB, Triton X-100, Tween-20 and Tween-80 is associated with a red shift of monomer absorption maxima at both pH values, suggesting that the micro-environment around mitoxantrone molecule is perturbed in comparison with phosphate buffer pH 7.4 and carbonate buffer pH 10. At pH 7.4, the red shift increases with surfactant in the order CTAB > Tween-80 > Tween-20 > Triton X-100 > SDS, whereas at pH 10 the red shift follow the order CTAB > Tween-80 = Tween-20 = Triton X-100 > SDS. This red shift indicates the transfer of mitoxantrone molecules from polar phase to a less polar phase in micellar medium. The octanol:water partition coefficient of mitoxantrone at pH 7.4 is logP = 0.79, which indicates that mitoxantrone is a fairly lipophilic drug [78
]. Therefore, in the presence of surfactant micelles mitoxantrone molecules prefer to move from polar aqueous medium in more hydrophobic medium of micelles.
Information about the position of mitoxantrone molecule in the micelle was obtained by comparing the absorption spectra of mitoxantrone in the presence of surfactant micelles with the spectra in water and solvents of different polarities. The absorption spectra of mitoxantrone recorded in protic solvents with different polarities indicated a red shift of both absorption maxima with the reduction of the solvent polarity. Also, the relationship between the position of monomer absorption maximum and the dielectric constant is linear at both pH values (Figure 6
Generally, these spectral shifts are interpreted as polarity changes of the immediate vicinity of the drug molecule [79
]. Therefore, the substitution of corresponding absorption maxima of monomer band for CTAB, SDS, Triton X-100, Tween-20 and Tween-80 micelles allowed one to determine polarity values corresponding to effective dielectric constants of 20, 58, 54, 49.5 and 40.5, respectively, for pH 7.4. For pH 10, the following effective dielectric constants were obtained: 3 for CTAB, 28 for SDS and 12 for Triton X-100, Tween-20 and Tween-80. It can be observed that for all surfactants, the dielectric constant at pH 10 is smaller than that at pH 7.4, indicating that at pH 10 when mitoxantrone molecule becomes uncharged and more hydrophobic penetrates deeper into micelles.
Since the micelle interface is an environment with a dielectric constant (ε = 36) [80
] intermediate between water (ε = 80) and 1,4-dioxane (ε = 2), mitoxantrone is encapsulated in CTAB and SDS micelles as monomer, and most probably situated in the micelle surface layer.
In the case of SDS micelles, the mitoxantrone chromophore ring is oriented towards the micelle core and both positively charged side chains oriented towards the negatively sulfate groups of SDS, both polar and electrostatic interactions playing important role in the drug-micelle binding [41
]. Also, this electrostatic attraction between opposite charges on mitoxantrone and SDS does not let the drug molecules penetrate deeply into the micelles. Therefore, the drug molecules are solubilized near the micelle surface.
In the case of CTAB micelles, the location of mitoxantrone molecules in the micelle surface layer can be explained by a cation-π interaction between the uncharged ring systems of mitoxantrone and the cationic head groups of CTAB, similar to the cationic dye pinacyanol [42
]. The lower spectral shift corresponding to a higher dielectric constant observed for the SDS micelles indicates that the micropolarity around mitoxantrone molecules in SDS micelles is different from that in CTAB micelles. In the case of positively charged CTAB micelles the electrostatic interactions between mitoxantrone molecules and micelles are absent and the hydrophobic effect prevails, resulting in a deeper penetration of mitoxantrone molecule into micelles (a higher spectral shift is observed, correlated with smaller dielectric constant). The flexibility of longer aliphatic chains of CTAB compared with SDS micelles can favor the easier movement of mitoxantrone molecules toward the core of CTAB micelles, resulting in a higher spectral shift [42
In the presence of non-ionic micelles, the monomer band is split into two components (spectra in Figure 2
]), which was not observed for SDS and CTAB micelles, but is more evident in the drug spectrum of mitoxantrone in 1,4-dioxane. Therefore, the new band around 647 nm, increasing with surfactant concentration, was assigned to the ionic-hydrophobic interactions of a part of the drug molecule present in a more hydrophobic medium [43
]. The palisade layer composed from the polyoxyethylene chains has a dielectric constant of 40–50 [82
] and the major part (band at 660 nm) of the drug molecule is most probably located in the palisade layer, which is known to be much thicker (25 Å) than the Stern layer of ionic micelles (6–9 Å) [42
], similar to the case of the antitumor antibiotic epirubicin [61