Mixed micelles as drug delivery nanocarriers

CHAPTER

Mixed micelles as drug delivery nanocarriers

9

´ Jan Sobczynski and Beata Chudzik-Rza˛d Medical University of Lublin, Lublin, Poland

CHAPTER OUTLINE 9.1 Physicochemical Basis for Mixed Micelles Formation .....................................331 9.1.1 Excipients ..................................................................................331 9.1.2 Micellization Process ...................................................................332 9.2 Mixed Micelles as Drug Delivery Nanocarriers ...............................................333 9.2.1 Excipients ..................................................................................333 9.2.2 Optimizing Micellar Properties .....................................................334 9.2.3 Mixed Micelle Formation ..............................................................335 9.2.4 Mixed Micellar Characterization ....................................................337 9.3 Solubilization of Drugs and Drug-Like Molecules in Mixed Micellar Systems ...338 9.4 Mixed Micellar Formulation for Antineoplastic Agents ....................................342 9.4.1 Micellar Delivery Systems in Cancer Therapy .................................342 9.4.2 Examples of Anticancer Formulations ...........................................345 9.5 Examples of Other Mixed Micellar Systems ....................................................352 9.6 Conclusions .................................................................................................356 References ..........................................................................................................356 Further Reading ...................................................................................................364

9.1 PHYSICOCHEMICAL BASIS FOR MIXED MICELLES FORMATION 9.1.1 EXCIPIENTS Mixed micelles used as drug delivery nanocarriers are often composed of block copolymers of different kinds, block copolymers mixed with amphiphilic molecules, or of block copolymers and classic anionic or cationic surfactants. The latter surfactants typically comprise a hydrophobic chain and a polar head group. While the anionic groups include carboxylates, sulfonates, and sulfates, the cationic ones are amines, quaternary ammonium halides and pyridine groups.

Design and Development of New Nanocarriers. DOI: http://dx.doi.org/10.1016/B978-0-12-813627-0.00009-0 © 2018 Elsevier Inc. All rights reserved.

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Copolymers for use in drug delivery systems typically include diblock or triblock copolymers, although some authors reported the use of pentablocks for drug delivery purposes (Fuentes et al., 2016; Parekh et al., 2016). The most common copolymers include ANA BNB, ANA BNB ANA and BNB ANA BNB type. NA and NB denote the degree of polymerization of the block monomers A and B and their values are usually as high as tens hundreds of units or more. For pharmaceutical applications, biodegradable polymers composed of biocompatible poly (ethylene glycol) (PEG) or poly(ethylene oxide) (PEO) blocks with low molecular mass (,B20 40 kDa; Webster et al., 2009) are preferred in order to enable renal clearance. A commonly used hydrophobic block is the poly(propylene oxide) (PPO). Considering the high amount of monomer units and sufficient difference between monomer type, these copolymers are intrinsically amphiphilic. Compared to small amphiphilic molecules, where the difference between chemical groups must be significant, the amphiphilicity of copolymers is achieved for less distinctive blocks. When matched with non-polymeric surfactants, the diblock copolymers may be treated as conventional surfactants composed of an alkyl hydrophobic chain and one hydrophilic head. While the triblock A B A copolymers are similarly built to bolaform surfactants, the triblock B A B copolymers structurally resemble gemini (dimeric) surfactants.

9.1.2 MICELLIZATION PROCESS In a selective solvent, where only one of the blocks dissolves, copolymers can form micelles through association of a few or numerous unimers. Amphiphilic diblock or triblock copolymers form micelles when isolated copolymer molecules (unimers) spontaneously assemble into nanosized aggregates above a certain concentration (critical micelle concentration, CMC) and temperature (critical micelle temperature, CMT) threshold. At these points the intrinsic solubility of amphiphilic unimers in aqueous media is exceeded. The CMC is often defined as the maximum attainable chemical potential of surfactant unimer and provides a convenient means to comparing properties of different surfactants. Despite the previously mentioned structural similarities, there are some important differences between polymeric and non-polymeric surfactants. Firstly, the alkyl chain and the head group are shorter than the respective hydrophilic and hydrophobic blocks of the individual polymer chains. Whereas the alkyl chain is short and virtually immobile, the hydrophobic block of the unimer with high NB may adopt a coil conformation upon exposure to water. In this conformation the contact between the aqueous phase and the hydrophobic block is largely prevented. Subsequently a monomolecular micelle is formed. Monomolecular micelles may not adsorb at the interfaces, due to lower energy state than the adsorbed state (Zana, 2005). At room temperature, the driving force enabling micelle formation is usually the release of water molecules adjacent to the hydrophobic block or moiety. The free hydrophobic groups may then form hydrogen

9.2 Mixed Micelles as Drug Delivery Nanocarriers

bonds with one another, resulting in an entropy gain. The micellar systems are typically composed of a hydrophobic micellar core and hydrophilic micellar corona. In contrast to classic surfactants, the hydrophobicity/hydrophilicity difference at the core/corona interface will not be as sharp for block copolymers. Whereas the alkyl or alkylaryl hydrocarbon chain of non-polymeric surfactants is highly and uniformly hydrophobic, the hydrophobic block of the copolymer often comprises an ether or ester oxygen, or amino group characterized with partial hydrophilicity. Consequently, the block will associate with water molecules and carry them into the micellar core upon micelle formation. The presence of the hydrophilic blocks on the micellar surface is responsible for repulsion forces stabilizing the micelle. However, the sterical repulsion provided by hydrated PEO blocks, for example, is smaller compared to the electrostatic repulsion of charged surfactants heads. Furthermore, there is a significant size difference between a charged surfactant polar head and a hydrophilic copolymer block. Thirdly, the copolymers exhibit some extent of polydispersity, resulting in a range rather than a single point CMC. Moreover, the values of CMC and CMT may not be easy to determine for block copolymers as they aggregate at low concentration.

9.2 MIXED MICELLES AS DRUG DELIVERY NANOCARRIERS 9.2.1 EXCIPIENTS The triblock PEO PPO PEO copolymers, Pluronics or Poloxamers, are characterized with complex structure. They have been extensively studied. They are often used in both basic research and formulation development studies. Owing to the variation in block length and the resulting different total hydrophobicity (hydrophilic-lipophilic balance (HLB) ranging from 1 to 29), Pluronics are very versatile polymers for use in drug delivery. They have been reported as efficient gene delivery agents as well as excipients for use in oral, ocular, and rectal drug delivery (Pitto-Barry and Barry, 2014). Based on distribution studies in model animals using Pluronic F68 and Pluronic P85, it was concluded that Pluronics are nontoxic agents that undergo renal excretion (Grindel et al., 2002a,b; Batrakova et al., 2004). The safety of numerous other Pluronics was also assessed (SinghJoy and McLain, 2008) and their biological properties will be discussed in detail in Section 9.4.1. Pluronics were reported to exhibit strong binding with compounds containing aromatic rings (Kozlov et al., 2000) and have shown a successful incorporation of a variety of different agents of different chemical structure (Alvarez-Lorenzo et al., 2011). The US Food and Drug Administration (FDA) approves eight Pluronics (L44, L61, L62, F68, F87, L101, F108 and F127) as pharmaceutical excipients (FDA, 2017).

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9.2.2 OPTIMIZING MICELLAR PROPERTIES The final size of the micelle depends on the unimers’ drive to minimize the entropy deficit associated with association of unimers into large structures. Chemical properties of the polymeric material and size of the polymeric blocks will influence the shape and size of micelle and the aggregation number (the number of monomers that form a micelle; Sawdon and Peng, 2013). The aggregation number is calculated by dividing the weight of the micelle by the weight of the individual unimer. A weight of individual micelle may be determined following light scattering or small-angle X-ray scattering measurements. It should be noted, however, that the aggregation number, micelle size, as well as shape are subject to change following an increase in concentration of the surfactant above CMC. Polymeric micelles are reported to be 5 100 nm in diameter. As the selfassembly of amphiphilic polymers is a reversible process, the polymeric micelles are characterized by their thermodynamic stability (potential for disassembly) and kinetic stability (rate of disassembly; Soo et al., 2009). While the thermodynamic stability depends on the length of the hydrophobic block, the factors governing kinetic stability also include the hydrophilic/hydrophobic block mass ratio, the physical state of the micellar core, and the presence of incorporated active pharmaceutical ingredient (API). Micelle stability may be increased by means of chemical crosslinking (R¯osler et al., 2001). Proper selection of the polymeric block enables development of an optimized formulation for the selected drug of interest. Every drug has a solubilization limit referring to temperature, nature and concentration of the surfactant. Large, asymmetrical, rigid molecules known to form crystals remain distinct within the micellar core, as they do not blend with surfactant hydrophobic part. On the other hand, compounds that are sparingly soluble in water and liquid at room temperature exhibit more flexible behavior (Liu et al., 2008). They will preferentially locate in the hydrophobic micellar core, where they mix freely with the coreforming block and subsequently form a homogenous mixture. Such solutes increase micelle size and influence the number of unimers forming the micelle. The aggregation number increases, following unimers’s drive to fill the swollen micellar core. The core of the copolymeric micelles may be large (depending on the NB) and/or be in the glassy state. Both properties are reported to increase the affinity between hydrophobic block and API. Solubilization within the core will also be influenced by the presence of water in the case of relatively polar block copolymers. For example, a hydrated oxypropylene region of the Pluronic F68 block may provide an insufficiently hydrophobic environment for apolar drugs, resulting in poor solubilization (Sobczy´nski et al., 2015). The amphiphilic molecules are expected to localize in the interface between the micellar core and the micellar corona. Such solutes will not influence the aggregation number, but may increase micellar size due to incorporation of solute molecules. The increased affinity is responsible for high drug entrapment efficacy and longer residence

9.2 Mixed Micelles as Drug Delivery Nanocarriers

time of the solubilized drug. Compared to classic surfactants, the copolymeric micelles may contain the solubilized API for a long time. Furthermore, low CMC assures that the content is not released by simple dilution in bodily fluids. The affinity may be increased by a chemical modification of the core-forming block, i.e., introduction of groups providing hydrogen bonding or π π interactions, rather than hydrophobic interactions or introducing a hydrophobic solute into the core (Zhao et al., 2011). Other approaches include chemical conjugation or complexation between API and a copolymer (Xiong et al., 2011; Miyata et al., 2011). The affinity between hydrophobic block and API will determine the drug binding strength within the polymeric micellar core and subsequently influence the release rate from a nanocarrier. Diffusion of API from micelles is influenced by the molecular weight (MW) of the API and the size of core-building block as well as API-micellar core affinity. Some additives may prove useful when formulating poorly soluble drugs in micelles. Studies performed by Hammad and Muller (1998) showed enhanced solubility of clonazepam in micelles enriched with alcohols possessing a phenyl ring. Moderately water-soluble alcohols decrease the CMC of copolymeric micelles and provide a good environment for clonazepam-possessing aromatic ring. In this respect the choice of benzyl alcohol or 2-phenylethanol as preservatives may be beneficial for formulation stability. Hydrophobic molecules trapped within a core characterized by high microviscosity will be slowly released, compared to molecules bound to micellar corona. Furthermore, micelle disintegration can be a result of a drug extraction, protein adsorption or protein penetration (Kim et al., 2010). Some nanocarriers are expected to release the API within the cellular compartments, while others are released at the vicinity of the targeted tissue (Judefeind and de Villiers, 2011). Drawbacks of using micellar drug delivery systems are associated with their small size, which limits their ability to carry a substantial dose of the active agent and may cause a premature release of the drug. Additionally, the synthesis of new polymeric materials, decorated with different chemical groups, e.g., homing devices enabling active targeting, increases the physicochemical complexity of the drug delivery system. Consequently, biocompatibility problems may occur (Kim et al., 2009, 2010).

9.2.3 MIXED MICELLE FORMATION Polymeric micelles formed from single copolymers often lack stability, exhibit unsatisfactory drug loading capacity, or broad size distribution, primarily due to limitations in the number of available building blocks. Therefore, mixed systems containing block copolymers of different kinds, block copolymers mixed with amphiphilic molecules, or of block copolymers and classic anionic or cationic surfactants have been proposed. Although binary surfactant mixtures have shown many advantages over single surfactant micelles, the thermodynamic properties of these systems are still not thoroughly explained. Commonly, new mixed micellar

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formulations are developed based on trial and error approach, requiring much experimental work. There is a need for comprehensive and predictive molecular theory that would broaden the understanding of the mixed micelles synergism. The theory would provide a scientific foundation for the development of new mixed micellar formulations and subsequently facilitate the optimization of new surfactant mixtures. In the binary surfactant systems, regular solution theory (RST) is often employed to characterize the micellar system. Mixing the two surfactants may result in ideal or nonideal mixtures. In terms of thermodynamics, the interaction parameter β is an energetic parameter representing the excess Gibbs free energy associated with surfactant mixing. The interpretation is correct, assuming the excess entropy of mixing equals zero, which is the assumption of RST. The β parameter accounts for the deviation from ideality, i.e., the more negative the β is, the more attractive interaction is observed. To the contrary, the positive β accounts for repulsive interactions. It should be noted that the obtained β values for many binary surfactant systems were found to vary, depending on solution composition. This suggests that the assumption of excess mixing entropy being zero may not be valid. Despite its limitations, the method is still very useful for studying mixed micellar systems as it provides a quantitative description of these systems. Owing to the well established methodology, the β parameters can easily be compared among different surfactant pairs. The attractive interaction between surfactants implies the synergism between the respective mixed micelle components. The synergism is observed when the CMC value for the binary system is lower than the CMC value of the respective single surfactant solutions. The latter will increase colloidal stability for mixed micellar system used in drug delivery. Molecular-thermodynamic theory refers to calculating the size and composition of mixed micelles, taking into account the free energy of the micelle-forming process. The latter is a sum of several free energy components described in this paragraph and an ideal entropy of mixing. The first transfer component relies on the behavior of free surfactant tails. The two tails are drawn from aqueous solution to micellar core, which is then similar to the binary oil mixture. The interfacial contribution represents the free energy change per surfactant molecule related to forming an interface between the micellar core and the surrounding aqueous medium. The packing contribution depends on the length of the surfactants forming micellar core. If there is a synergism between their hydrophobic parts, the surfactant will better fill the micellar core and provide more negative free energy input. The steric contribution depends solely on the alignment of polar groups at the micellar interface. The latter is associated with the size of head groups or polar blocks. In spherical micelles, a larger area per surfactant molecule leads to lower sterical contribution. The final electrostatic contribution depends on the coulombic interactions of the polar head groups. Considering a charged surfactant is mixed with a nonionic surfactant, the charged heads become diluted within the

9.2 Mixed Micelles as Drug Delivery Nanocarriers

micellar corona and charge-bearing groups are further apart. Consequently the same-charge repulsion is reduced and micelles form more easily. Micelles may form spontaneously in aqueous media upon dissolution of pure surfactants, as the micelle formation is an entropy-driven process. However, sometimes an input of energy is required when the drug of interest is mixed with pure surfactants. The transfer of the drug into micellar core will require more laborious handling. The latter usually involves shaking or vortexing the medium. Sometimes the significant energy must be provided in the form of probe or bath sonication. There are also literature examples of using solvent evaporation method. The components of formulation are then dissolved in an organic solvent, mixed, and the solvent is evaporated. The residue forms a thin film which is then dissolved in water.

9.2.4 MIXED MICELLAR CHARACTERIZATION In order to depict the synergism of the surfactant micellization, an experimental determination of mixed micellar CMC is needed (see Sections 9.1.2 and 9.2.3). As previously described, this preformulation step will lead to choosing the most promising surfactant pair with regard to thermodynamic stability of the mixed nanomicellar system. The CMC is commonly developed by surface tension measurements, e.g., using Wilhelmy plate technique. The surface tension will decrease upon addition of higher surfactant mixture content. The break at the surface tension vs concentration curve denotes the CMC value. The CMC value can also be determined by the iodine solubilization method, the pyrene probe solubilization, and the dye solubilization method. The methods are based on the difference in fluorescence emission spectra of the solubilized agent in the micellar core and bulk aqueous phase. When concentration of the micelle-forming agent reaches the CMC, the agent becomes solubilized, yielding a sharp increase in fluorescence intensity or a shift in fluorescence maximum. Clouding behavior is a physical change occurring in homogenous solutions of amphiphilic substances. The solution becomes separated into a surfactant-rich and surfactant-poor phase at a defined temperature, called cloud point (CP). The reason for the phenomenon is the dehydration of hydrophilic portion of nonionic surfactants at higher temperature. The CP is often measured to characterize nonionic surfactants. At increasing temperature, micelles attract each other and may form large aggregates, indicating formulation instability. The CP will be affected by inter-micellar interactions and is thus a valuable property to evaluate mixed systems. Brownian movements of particles in aqueous environments are the basis of particle size measurements by means of the dynamic light scattering (DLS). The particle characteristics are derived from an autocorrelation of the intensity trace recorded during the experiment. While the lower limit for the size measurements is low (0.3 nm), the size of micellar nanovesicles is not expected to exceed the

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upper limit (a few micrometers). The method enables analysis of nanoparticles in aqueous environments, even when different materials are present in one sample (Figueiredo, 2013). The disadvantages of DLS is that small particles are not detected in the presence of larger entities. It is recommended to present the results using the intensity mode and give the average polydispersity index acquired in 3 10 measurements (Hackley and Clogston, 2011). The refractive index (n) of the material and the viscosity of the medium has to be known before performing DLS measurements. As the particle diameter and polydispersity is crucial with respect to efficient drug delivery (see Section 9.4.1), another technique should be used to further prove the DLS results. The most widely reported supplementary techniques are based on electron microscopy, namely transmission electron microscopy (TEM) and its modification, cryo-TEM. Due to the fact that the analyzed particles’ shape often deviates from spherical, TEM may provide additional benefits. Electron microscopy reveals the nanocarrier morphology, showing particle shape, roughness and irregularities (Hwang et al., 2009). In the zeta (ζ) potential measurements, the electrophoretic mobility in an electrical field applied across the sample of nanoparticulate formulation is measured by laser Doppler velocimetry. The ζ-potential will influence particle aggregation as well as the nanoparticle’s ability to permeate biological membranes, the mechanism of cell entry, and t1/2 in vivo. It is therefore a critical parameter with regard to formulation stability and cell internalization (Iversen et al., 2011).

9.3 SOLUBILIZATION OF DRUGS AND DRUG-LIKE MOLECULES IN MIXED MICELLAR SYSTEMS As described in previous sections, mixed micelles exhibit enhanced thermodynamic stability, compared to pure nonionic micelles. Moreover, the addition of another surfactant may serve to control micellar size and surface charge (Fig. 9.1). Mixtures of block copolymer yield larger micelles, compared to micelles formed by charged surfactants alone. Furthermore, block copolymers with high binding affinity to API may be introduced in order to increase drug loading. Consequently, the capability of these systems with regard to solubilization of poorly watersoluble drugs increases. Bhat et al. (2009) determined that negatively charged, poorly water-soluble drug naproxen is efficiently solubilized within micellar corona of mixed, positively charged micelles. The negatively charged, hydrophobic drug was efficiently retained via hydrophobic and ionic interactions. Mondal et al. (2016) studied the encapsulation of drug-like molecule, phenosafranine (PSF), by mixed micelles composed of sodium dodecyl sulfate (SDS) and Pluronic F127 (Fig. 9.2). The binary mixture of 2 mM F127 and 2 mM SDS yielded 8 nm nanovesicles, compared to 22 nm formed by pure F127. The

9.3 Solubilization of Drugs

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FIGURE 9.1 Size distribution for plain micelles of Pluronic P123 (1%; black line), plain micelles of Hydriosul KN.40 (sodium laureth-30 sulfate; 20%; dotted line) and mixed micelles Pluronic P123:Hydriosul KN.40 1%:10% (55 ), 2%:1% (gray line) and 0.2%:1% (- - -) unpublished results.

FIGURE 9.2 Schematic representation of mixed micelles composed of Pluronic block copolymer F127 and the anionic surfactant sodium dodecyl sulfate (SDS) with encapsulated PSF. Reprinted with permission from Mondal, R., Ghosh, N., Mukherjee, S., 2016. Enhanced binding of phenosafranin to triblock copolymer F127 induced by sodium dodecyl sulfate: a mixed micellar system as an efficient drug delivery vehicle. J. Phys. Chem. B 120(11), 2968 2976. Copyright 2014 American Chemical Society.

formation of mixed micelles was driven by hydrophobic interactions, as shown by large positive enthalpy and entropy changes. When PSF binds to the F127 or mixed micelles the entropy and enthalpy are positive, highlighting the role of hydrophobic forces in PSF binding. Steady-state absorption and fluorescence emission studies revealed a blue shift of PSF emission maximum and red shift of

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absorption maximum. The phenomena were explained by the fact that, in mixed micelles, PSF was incorporated in more hydrophobic environment compared to plain F127 micelles. Further steady-state fluorescence anisotropy measurements and isothermal titration calorimetry studies showed that PSF was bound more strongly within mixed micelles compared to F127 micelles. The potassium iodide quenching studies showed that PSF is deeply embedded in mixed micellar systems. The time-resolved fluorescence anisotropy data indicated that in mixed micellar system PSF molecules were predominantly bound to F127, while encapsulation of F127 yields only B55% PSF binding. Upon adding SDS, almost all PSF molecules were drawn inside the mixed micelles from the bulk aqueous medium. PSF mobility was also more restricted, as shown by analysis of PSF fluorescence quantum yields in mixed micellar system. Thummar et al. (2011) studied the interaction between linear surfactants, dodecyltrimethylammonium bromide (DTAB) and SDS, with Triton X-100 (TX100) and Pluronic P105 by means of DLS as well as viscosity and surface tension measurements. The authors observed a cooperative binding of surfactants and copolymeric chains at low surfactant concentration. Higher surfactant content pushes the P105 unimer-micelle equilibrium to the unimer side. While the binding between SDS and Triton X-100 was strong, the binding of DTAB was weaker due to partial repulsion between the protonated ethylene oxide block and cationic DTAB. An increase of linear surfactants concentration in P105-surfactant binary mixtures led to a decrease of P105 hydrodynamic radius of P105 micelles. The decrease reached a pseudo-plateau value at higher surfactant mole fraction. The molar ratios for the plateau values decreased in the order TX100 , SDS , DTAB. While DTAB and SDS exhibited strong tendency to dissociate P105 micelles into unimers, TX-100-P105 mixed micelles resisted breakdown. The increase of intrinsic viscosity upon increasing surfactant concentration was explained by the fact that copolymer-surfactant micelles were more hydrophilic, compared to plain P105 micelles. The binary systems were further used to solubilize anti-inflammatory steroid drug, dexamethasone. The hydrophobic dexamethasone showed high solubility in P105 micelles, compared to water and surfactant-P105 binary mixtures. This was explained by the surfactant tendency to dissociate P105 micelles. Accordingly, the release of dexamethasone from micellar systems incorporated into agar gels was higher for binary P105surfactant systems compared to plain P105 micelles. The linear surfactants could serve as a release modifiers in such systems. Kumar et al. (2016a) tested combinations of various positively charged surfactants to optimize a nanomicellar formulation of curcumin. The following surfactants were used in the study: dodecylethyl dimethylammonium bromide (DDAB)—as well as a series of double chain surfactants with number of carbon atoms ranging from C12 to C18, didodecyl dimethylammonium bromide, ditetradecyl dimethylammonium bromide, dihexadecyl dimethylammonium bromide, and dioctadecyl dimethylammonium bromide. Firstly, critical aggregation

9.3 Solubilization of Drugs

concentration (CAC) values for each DDAB:double chain surfactant pair (1:1) were obtained. This was done by carrying out conductivity and fluorescence measurements at increasing surfactant concentration. CAC was directly dependent on the number of carbon atoms in the double chain surfactants, i.e., the highest value was obtained for didodecyl derivative 1 DDAB pair and the lowest for dioctadecyl derivative 1 DDAB pair. The hydrodynamic diameter of the obtained nanomicelles was further measured showing a trend of decreasing diameter with increasing number of carbon atoms (except for dioctadecyl derivative). The average size ranged from 174.2 to 303.8 nm with high polydispersity index (PDI) (0.293 0.510). Mixing of double chained surfactant with a single chained one leads to DDAB penetration into the outside layer of vesicle bilayer. Subsequently, an asymmetry arises causing the bilayer to crack. The bilayer is further reorganized, leading to smaller aggregates. Solubility of curcumin encapsulated within mixed micellar formulations increased in the order of 103 to 104 depending on the formulation and a small red shift was observed in curcumin fluorescence emission spectra. In general, the more carbon atoms in the double chain surfactant, the better solubilization was achieved. In addition, an exceptionally high solubility was achieved for DDAB:didodecyl derivative, due to enhanced stability of aggregates resulting from chain length symmetry. Curcumin location in nanomicelles was further examined by absorbance spectra peak shifts and separations fluorescence studies (for polar regions sensing) and fluorescence quenching experiments by use of potassium iodide and acrylamide. Curcumin molecules were found near the polar head groups of vesicles. Curcumin-loaded nanomicelles were much smaller, i.e., 15 18 nm and more compact compared to blank carriers. The degradation of curcumin in alkaline pH was significantly suppressed (60% 75%) upon encapsulation in mixed micelles. Curcumin ability to scavenge free radicals, which is responsible for some biological activities, was enhanced 2 3 times. Parmar et al. (2014) studied the influence of alkyl trimethylammonium bromides with varying number of carbon atoms (C10, C12, C14 and C16) on their assembly with hydrophobic Pluronics P103, P104 and P105. Mixed micellar carriers were assembled with micellar core formed by PPO blocks, and alkyl hydrophobic chains of charged surfactants and micellar corona, comprising PEO blocks and charged surfactant head. This arrangement generated repulsion between Pluronic micelles and hindered dehydration of PEO units. The addition of surfactants induced repulsion between PEO blocks. Subsequently, Pluronic aggregation with temperature was reduced, as shown by CP measurements. This effect was especially visible when C16 surfactant was used, while the addition of C10 surfactant did not lead to drastic changes in CPs. As a result of incorporating surfactant molecules into Pluronic micelles, authors observed a reduction in the hydrodynamic micellar radius. Surfactants with longer aliphatic chains yielded stronger reduction, compared to their short-chain counterparts. Furthermore, authors solubilized hydrophobic drug hydrochlorothiazide into mixed micelles composed of P103 and C14 trimethylammonium bromide. At alkaline pH ( . 10) hydrochlorothiazide exists as

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an anion. The presence of cationic surfactant in mixed micelles neutralized the charge on hydrochlorothiazide and thus facilitated its solubilization. For aqueous solutions of P103, the micellar number density increased with increasing concentration and thus more drug molecules could be solubilized into the micellar phase. In mixed micellar systems, even more drug loading could be achieved by incorporating more surfactant molecules and modulating formulation pH. Kumar et al. (2016b) aimed to solubilize curcumin in mixed micellar systems composed of cationic and nonionic surfactants. The following surfactants were used: DDAB, Brij 96 (polyoxyethylene (10) oleyl ether), Tyloxapol, Tween 80. The CMC studied by means of fluorescence and conductivity data showed that mixed micelles composed of Tween and DDAB yielded the smallest CMC, compared to mixed systems containing other surfactants. Curcumin solubilized in mixed nanomicelles showed a significant blue shift of the fluorescence emission spectrum from λ 5 550 to 500 nm, compared to aqueous medium. The fluorescence quenching experiments with potassium iodide revealed curcumin localization in the partially polar microenvironment at head group of the charged surfactant. The fact was confirmed by TEM imaging. The microscopic pictures showed curcumin molecules at outer micellar regions, outside of the micellar core. The size of nanomicelles was within 30 40 nm range and increased upon curcumin encapsulation, further proving the location near the head groups. While enolic and phenolic groups of curcumin exhibit electrostatic interactions with positively charged head group surfactants, the hydrocarbon group of curcumin is embedded within the medium of the micelles. The effectiveness of curcumin solubilization expressed as curcumin partition coefficient between micellar and aqueous phase was calculated based on analysis of UV absorption spectra. Brij 96/DDAB exhibited the highest solubilization, probably due to good compatibility between linear structure of Brij 96 and linear hydrophobic chain of DDAB. The other nonionic surfactants used in this study have more bulkier heads and form distorted micelles, leading to less efficient curcumin solubilization. The stability of curcumin in alkaline pH was suppressed by encapsulation in mixed micellar systems. DDAB/ Tyloxapol and DDAB/Brij 96 systems where the most efficient curcumin stabilizers in this regard. 2,2-Diphenyl-1-picrylhydrazyl test was used to assess curcumin free radicals scavenging ability in mixed micelles. The ability was remarkably enhanced (2 3x) in mixed micelles, compared to plain curcumin solution.

9.4 MIXED MICELLAR FORMULATION FOR ANTINEOPLASTIC AGENTS 9.4.1 MICELLAR DELIVERY SYSTEMS IN CANCER THERAPY Many mixed micellar formulations are being developed for cancer treatment in order to overcome problems associated with current formulations for antineoplastic agents. Some of the drawbacks of currently used therapeutic agents include: lack

9.4 Mixed Micellar Formulation for Antineoplastic Agents

of specificity and subsequent toxicity to normal tissues, unfavorable biodistribution and pharmacokinetic properties, inability of drugs to cross physiological barriers due to high interstitial fluid pressure in tumors, and compromised and anisotropic blood supply. It has been shown that nanocarriers are characterized by interesting and new features, useful in drug delivery of antineoplastic agents, allowing to overcome pharmaceutical problems associated with currently used intravenous dosage forms (e.g., solubility, stability) (Cooper, 2010). Use of nanovehicles allows for targeted drug delivery, overcoming biological barriers and addressing the problem of multidrug resistance (MDR; Malam et al., 2011). Moreover, nanoparticulate drug delivery systems offer protection against chemical and enzymatic degradation, prolong drug clearance, and decrease drug interactions with nontargeted tissues leading to decreased drug toxicity. Moreover, two antineoplastic agents can be present in one nanocarrier, facilitating combination drug therapy (Magadala et al., 2008; Khdair et al., 2010). Various polymers can be employed optimizing the formulation of the specific antineoplastic agent, delivery route, and site (Xiong et al., 2009). An outer hydrophilic shell, usually composed of PEG or PEO will hinder interactions with proteins and cell surfaces—the “stealth” functionality (Li and Huang, 2010; Ikeda and Nagasaki, 2012). The micelles likely avoid extravasation, leading to decreased side effects of incorporated API, as normal vessels interendothelial junctions only allow for passage of entities of 6 7 nm (Hobbs et al., 1998). The hydrophilic micellar surface prevents particle aggregation as well as opsonization in bloodstream, and subsequent clearance by mononuclear phagocytic system. As a result, half-life following intravenous administration is increased. Copolymeric micelles exhibit prolonged circulation time in vivo, half-life (t1/2) up to 19 h, as determined in rodents (Rolland et al., 1992; Yamamoto et al., 2001; Lukyanov et al., 2002; Lukyanov et al., 2004). The size, shape and surface properties of particles influence the efficiency of internalization into cancer cells (Lee et al., 2010a; Cabral et al., 2011) as well distribution within the cell (Lee et al., 2010b). Cell internalization mechanisms include merging with cellular membranes or absorption by endocytosis (Torchilin, 2009; Horobin, 2010; Sahay et al., 2010). 100 nm in diameter was found as an optimal size with regard to efficient cell internalization (HarushFrenkel et al., 2008). While the small, uncharged particles ,25 nm will be rapidly cleared through kidney glomeruli, large particles ( . 200 nm) may be sequestrated into spleen (Moghimi and Hamad, 2009). The preferential accumulation of macromolecular API and nanovehicles in cancer tissues is referred to as the enhanced permeability and retention (EPR) effect, described by Matsumura and Maeda (1986). Blood vessels in the vicinity of the solid tumors exhibit erratic and bidirectional blood flow, increased vascular density and permeability, lack of smooth muscle lining, and subsequent lack of angiotensin II-induced vasoconstriction (Vaupel, 2012). Vascular architecture is tortuous and irregular due to chaotic angiogenesis. The EPR effect allows for accessing the area of intensive cancer growth through gaps and fenestrations in endothelial layer of tumoral microvessels (Chauhan et al., 2012; Maeda, 2012). Slow venous return, as well as impaired

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lymphatic drainage, leads to accumulation of proteins, macromolecular drugs, polymeric and lipidic particles from the interstitium of tumor (Maeda et al., 2009; Acharya and Sahoo, 2011). Drug delivery nanoparticles, depending on the material characteristics (e.g., shape, surface characteristics, deformability), exhibit EPR effect if their size corresponds to the diameter of the cancer microvessels (100 780 nm; Dreher et al., 2006). Nanoparticles of intermediate size (10 100 nm) can achieve extended residence time in the blood by minimizing accumulation in the liver and spleen (Ekdawi et al., 2013; Yhee et al., 2013). The increased circulation time will further enhance the retention simply by increasing exposure of the nanovehicle to the tumor (Perrault et al., 2009; Wang and Thanou, 2010). Release of the API can also be stimulated by external application of ultrasound or increased temperature applied in the vicinity of the solid tumor, providing even more selective treatment (Talelli and Hennink, 2011; Rabanel et al., 2012). The selectivity of a nanocarrier towards a specific type of cancer cell can be improved by use of active targeting, which is based on attaching a recognition unit (e.g., a sugar, an antibody, an aptamer, a peptide or a small molecule; Zhang et al., 2011; Holgado et al., 2012). Polymeric micelles can be then functionalized with homing devices to allow more selective and efficient therapy by active targeted drug delivery (Huang et al., 2012). Passive targeting is based on the distinguished physical properties of cancer tissues, including the size of the tumor tissue fenestrations, reduced tumor pH, and different redox potential (Deng et al., 2012; Saravanakumar and Kim, 2014) compared to normal tissues. For intravenously injected formulation, a pH 7.4 is maintained when it is contained within the systemic circulation. However, when micelles become sequestrated into cancer extracellular matrix via EPR effect, they would encounter a more-acidic pH (as low as 5.5, Yin and Bae, 2012). A pH-responsive carrier will be stable in neutral pH, but undergo a structural rearrangement and subsequent release of the loaded drug. Two major approaches have been proposed in this regard. Firstly, a “titratable” polymeric block that contains weakly basic dissociates upon protonation of N atoms and turns from hydrophobic to hydrophilic. Following the hydrophilicity increase, the micelle is rapidly dissolved, leading to drug release. Secondly, the polymer bears an acid-cleavable linkages. The linkage can be placed in various parts of the block and either connect the block with the active agent, or be an integral part of the block. Upon contact with acidic pH micelle, can thus either completely disintegrate or leak API in a controlled manner. Some copolymers additionally display a range of unique biological effects. Hydrophobic and amphiphilic Pluronic unimers are efficiently internalized by cancer cells and decrease the cellular membrane microviscosity. Furthermore, Pluronics may decrease the number of lysosomes and their acidity, by interaction with ATP-dependent proton pump (Venne et al., 1996; Alakhov et al., 1999; Miller et al., 1999; Rapoport et al., 2002). An inhibition of cellular respiration, following Pluronic translocation into mitochondrial membranes, increases the

9.4 Mixed Micellar Formulation for Antineoplastic Agents

production of ROS and a subsequent release of cytochrome c, contributing to enhanced doxorubicin-induced apoptosis. Pluronics have been demonstrated to enhance API accumulation in the susceptible cell nucleus. Subsequently, Pluronics would increase the efficiency of some chemotherapeutic agents. Micelles composed of Pluronics were proven effective against MDR cancer cells, by inhibiting glycoprotein P mediated drug efflux (Wei et al., 2013). A Pluronicinduced ATP depletion may also enhance intestinal drug transport (Guan et al., 2011) and enable the API to cross the blood brain barrier.

9.4.2 EXAMPLES OF ANTICANCER FORMULATIONS Abouzeid et al. (2014) used mixed micelles composed of PEGphosphatidylethanolamine (PEG2000-PE) and vitamin E for co-delivery of curcumin and paclitaxel. The micelles were prepared by the thin film hydration method, reaching B90% drug loading efficiency for optimized drug doses. The formulation exhibited only 10% burst release, followed by a slow release of the remaining load. The drug loads, as well as micellar size and zeta potential, were retained for at least 2 months at 4 C. Further activity of the mixed micellar formulation was evaluated using normal (SKOV-3) and paclitaxel-resistant (SKOV-3TR) human ovarian carcinoma monolayer cultures. Even though the addition of curcumin did not increase the cytotoxicity of paclitaxel in vitro against SKOV-3 cell line, a synergistic effect was observed in the case of the SKOV-3TR line. This was explained by the fact that curcumin enhanced paclitaxelinduced cytotoxicity by downregulating nuclear factor NFκB and the Akt pathways. The formulation was also administered intravenously in SKOV-3 and SKOV-3TRbearing mice. The combination treatment exhibited superior tumor inhibition properties in both normal and paclitaxel-resistant groups, suggesting that curcumin reversed MDR. The combination treatment was successful at inhibiting tumor growth threefold, compared to treatment with paclitaxel alone (Table 9.1). Chen et al. (2015) addressed the issue of a concomitant delivery of a hydrophilic and hydrophobic antineoplastic agents by developing mixed micelles based on doxorubicin-Pluronic P105 conjugate (Dox-P105) and Pluronic F127. The Dox-P105/F127 micelles were loaded with hydrophobic paclitaxel at a copolymer/drug ratio 10:1:1, following thin film hydration method. The in vitro release profiles showed fast drug release at pH 5.0 in contrast to sustained release at pH 7.4. The encapsulated paclitaxel was released via hydrophilic channels from the micellar core into Tween 80 enriched medium. However the doxorubicin was released by a gradual hydrolysis of the amino bond of P105-Dox conjugate, as shown by addition of cathepsin B to the release medium. The in vitro cytotoxicity was evaluated using normal breast cancer cell line MCF-7 and multidrug resistant κB cells. The synergistic effect of drugs and mixed micelle components suggested that Pluronics may act as chemosensitizers and potentiate the cytotoxic effect in multidrug resistant cancers. Confocal laser scanning microscopy imaging showed

345

Table 9.1 Examples of Anticancer Mixed Micellar Formulations

Authors

Encapsulated Molecule and Mixed Micelle Material

Dahmani et al. (2012)

Paclitaxel, (1) F127/LHR; (2) F68/LHR

Abouzeid et al. (2014)

Curcumin and paclitaxel PEG-PE/vitamin E (89:11 molar ratio) micelles

Chen et al. (2015)

Paclitaxel, P105-Dox/F127

Dou et al. (2014)

Docetaxel, (1) MPP/TPGS; (2) MPP/TPGS/CSO-SA

Zhang et al. (2014)

Transhinone IIa, TPGS-gPLGA/F68 (3.2:1)

Zhao et al. (2012)

Curcumin, Pluronic P123: Pluronic F68 (2.05:1)

Zhao et al. (2014) Chen et al. (2013)

Paclitaxel, Pluronic L121: thiolated Pluronic conjugate (8:2, w/w) Docetaxel, Pluronic P105: Pluronic F127

Bernabeau et al. (2016)

Paclitaxel, soluplus/TPGS (4:1)

Sheu et al. (2016)

Docetaxel (1 mg/g), Pluronic F127:Pluronic L121 (15 mg/ g:45 mg/g)

Characteristics of the System Blank: CMC (1) 0.049 mg/mL; (2) 0.055 mg/mL Loaded: diameter (1) 135.5 6 0.5; (2) 131.2 6 2.2 nm; PDI (1) 0.135; (2) 0.177 Blank: 15.6 6 1.9 nm diameter zeta potential 227.7 6 .7 mV; CMC 1.66 3 10 5 M Loaded: 19.3 6 1.9 nm diameter Blank: CMC 0.0063%; 28.76 nm diameter Loaded: 22.26 6 0.34 nm diameter (PDI 5 0.14), ζ-potential 21.48 6 0.19 mV Blank: CMC (1) 5.51 3 1025 M; (2) 2.11 3 1025 M Diameter (1) 30.7 6 1.13 nm; (2) 44.96 6 1.82 ζ-Potential (1) 2.32 mV; (2) 27.12 mV Loaded: diameter (1) 21.03 6 1.18 nm; (2) 34.96 6 0.51 nm ζ-potential; (1) 6.69 mV; (2) 29.87 mV Blank: 121.5 6 2.5 nm diameter, ζ-potential 28.46 6 0.52 mV, CMC 5.011 mg/L Loaded: 143.6 6 5.7 nm diameter, ζ-potential 216.43 6 1.42 mV Blank: 23.4 nm diameter (PDI 5 0.197), ζ-potential 21.7 mV, CMC: 1.86 3 1025 M Loaded: 68.2 nm diameter (PDI 5 0.304), ζ-potential 28.46 6 0.52 mV Blank: CMC 0.038 μg/mL Blank: 20.2 nm diameter, CMC 7.61 3 1026 M Loaded: 23 nm diameter Blank: CMC 0.0042 mM Loaded: 119.7 6 5.6 nm diameter, PDI 0.122 Loaded: 215.6 6 11.5 nm diameter, ζ-potential 235.9 6 4.9 mV

CSO-SA, stearic acid grafted chitosan oligosaccharide; LHR, heparin all trans retinoic acid; MPP, monomethylol poly(ethylene glycol)-poly(D,L-lactic acid); TPGS, D-α-tocopheryl polyethylene glycol 1000 succinate.

9.4 Mixed Micellar Formulation for Antineoplastic Agents

that internalization of mixed micelles occurring via an energy-dependent endocytosis, involving both clathrin- and caveolae-mediated mechanisms. Following the uptake, doxorubicin was released in lysosomes as a result of enzymatic cleavage and bound to cell nuclei where it exerted its pharmacological action. Annexin VAPC/7-AAD staining based quantitative apoptosis measurements showed higher apoptosis rates for cells treated with paclitaxel-loaded Dox-P105/F127 micelles compared to drugs alone, owing to the intrinsic biological activity of Pluronics. Moreover, the mixed micellar formulation induced a significantly better S-phase arrest compared to simultaneous use of doxorubicin and paclitaxel. An anti-tumor efficacy of mixed micelles was further evaluated in MCF-7/ADR spheroids and subcutaneous xenograft mouse model. The mixed micellar formulation yielded the most significant suppression of tumor proliferation. The administration of mixed micelles in tumor-bearing mice yielded lower drug accumulation in liver, spleen and heart, the latter being especially relevant in respect to documented doxorubicin cardiotoxicity. On the contrary the drug concentrations were found higher in plasma and tumor, probably due to the enhanced permeability and retention effect. Chen et al. (2013) used Pluronic P105 and Pluronic F127 mixed micelles and further loaded them with docetaxel. The micelles were obtained using thin film hydration and subsequent lyophilization. The authors used central composite design to optimize fabrication parameters. The differential scanning calorimetry (DSC) thermogram confirmed the entrapment of docetaxel within the hydrophobic micellar core. Whereas the free drug could freely dissolve through dialysis membrane, the dissolution of docetaxel from mixed micellar formulation exhibited an initial burst release during first 2 h followed by a continuous release, fitting the Weibull model. The formulation showed high storage stability, as 95.7% of the drug was preserved during 6 months storage at 4 C. The cytotoxic efficacy was further tested using human alveolar basal epithelial adenocarcinoma cell line A549 and its paclitaxel-resistant subclone. Whereas in A549 cell line the activity of commercial docetaxel preparation and mixed micellar formulation was similar, the latter exhibited a hypersensitization effect in paclitaxel-resistant line. In vivo pharmacokinetic studies revealed that encapsulation of docetaxel led to nearly four times increase of area under the curve (AUC) and an increase of t1/2 of docetaxel in rats. The tumor inhibition rate in rats was higher for mixed micellar formulation compared to group treated with commercial docetaxel formulation, Taxotere. Moreover, the extent of weight loss of animals treated with mixed micellar docetaxel was lower than that induced by Taxotere. Zhao et al. (2014) aimed to improve paclitaxel oral bioavailability by preparing mixed micelles composed of Pluronic L121 and a thiolated Pluronic conjugate. Firstly, hydroxyl groups of Pluronic F127 were conjugated with carboxylic groups of poly(acrylic acid) (PAA). Secondly, the carboxylic groups of PAA were conjugated with primary amino groups of L-cysteine yielding pH-reactive

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F127-PAA-cysteine copolymer (pKa 5 4.8). TEM imaging showed that mixed L121/F127-PAA-cysteine micelles were round and their mean diameter was between 10 and 120 nm. The micelles were further loaded with paclitaxel with drug loading of 2.87 6 0.23%. The mixed micellar formulation displayed a delayed drug release in vitro. Owing to the deprotonation of PAA, only 20% of paclitaxel was released in simulated gastric fluid over 2 h. Paclitaxel absorption studies in excised rat intestines proved paclitaxel-loaded micelles absorbed via passive transfer by diffusion across membranes. The permeation was similar in duodenum, jejunum, ileum, and colon and was not influenced by the addition of P-glycoprotein efflux pump (Pgp) inhibitor, verapamil. The pharmacokinetic parameters were determined in rats for mixed micellar formulation and plain paclitaxel solution. The AUC nearly quadrupled and the t1/2 increased 1.6 times as a result of formulating paclitaxel in mixed micelles. Authors attributed the increase paclitaxel oral bioavailability to Pluronic inhibition of Pgp efflux pumps and prolonged residue time at the site of adsorption. Zhang et al. (2014) encapsulated tanshinone IIa (TAN), a potent phenanthro [1,2-b]furan-dione derivative, in mixed micellar system to enhance its antineoplastic activity. Firstly, the copolymer of D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) and poly(D,L-lactide-co-glycolide) (PLGA) was prepared using chemical graft copolymerization. The TPGS-g-PLGA and Pluronic F68 were used to prepare mixed micelles by film hydration method. In order to optimize preparation conditions, central composite design/response surface methodology was utilized. The optimized condition included 41 C process temperature, 3.2:1 TPGS-g-PLGA to F68 ratio, as well as 14:1 polymer to drug ratio. The measured micellar size and zeta potential remained stable over a period of 7 days in phosphate buffered saline and when exposed to bovine serum albumin over 48 h. The mixed micellar formulation exhibited a slow in vitro release with only 20% and 50% of the drug released over 8 and 96 h, respectively. Further tests were performed in human liver carcinoma HepG2 cells. The TAN encapsulated in mixed micellar nanovesicles increased cell apoptotic rate, compared to free drug, as shown by morphology observations, mitochondrial dysfunction measurement, Annexin V/PI dual staining, and examination of pro-apoptotic proteins. Mixed micelles loaded with fluorescent probe nile red were efficiently taken up by the cells as shown by confocal laser scanning microscopy imaging and flow cytometry. Although the cmax of TAN was higher for plain TAN solution, the mixed micellar formulation yielded three times higher AUC, following administration in male Wistar rats. Zhao et al. (2012) prepared mixed micellar formulation of Pluronic P123 and Pluronic F68 by thin film hydration method. The authors used central composite design to optimize fabrication variables. At optimized conditions, the solubility of curcumin reached 3.02 mg/mL compared to 11 ng/mL in water. Only 10% of curcumin was released in vitro from micellar formulation within first 6 h, compared to 66% released from propylene glycol solution. The in vitro cytotoxicity studies

9.4 Mixed Micellar Formulation for Antineoplastic Agents

in MCF-7/ADR cells revealed higher cytotoxic effect in drug-resistant line compared to drug-sensitive cells. Dou et al. (2014) aimed to increase docetaxel bioavailability following oral administration. The system comprised monomethylol poly(ethylene glycol)-poly (D,L-lactic acid) (MPP), D-α-tocopheryl polyethylene glycol 1000 succinate and stearic acid grafted chitosan oligosaccharide (CSO-SA). The latter was produced by coupling reaction between carboxyl groups of stearic acid and primary amino groups of chitosan. The study aimed to find the right balance between favorable excipients. TPGS provided good thermodynamic stability to the micelles and could protect docetaxel from Pgp efflux; CSO-SA accounted for good biocompatibility, biodegradability and mucoadhesive properties; and MPP bound docetaxel provided good entrapment efficiency. Previous preformulation studies showed that CSO-SA had minor impact on docetaxel solubilization in micelles. Two types of mixed MPP-TPGS micelles were produced by thin film hydration method, i.e., with and without CSO-SA, loaded with docetaxel and characterized. The increase of zeta potential upon implementing CSO-SA was observed, due to cationic groups of chitosan. Over 1 week, 99% of drug was retained in micelles showing good formulation stability. The in vitro release of docetaxel was studied using simulated gastric fluid (1 h) and simulated intestinal fluid (48 h). The MPP core retarded the diffusion of water into micellar core and subsequently micelle disintegration, producing a sustained docetaxel release. The slow release could also be caused by the extensive swelling of CSO-SA. Pharmacokinetic studies in rats showed a 2 3 and 3 3 increase of AUC for micelles without and with CSO-SA, respectively. Deliver docetaxel (DTX) solution administered orally served as a control formulation. The in vivo results showed faster drug release compared to in vitro conditions. However, bioavailability enhancement was explained by the presence of bile acids and digestive enzymes that contributed to micelle degradation. Dahmani et al. (2012) prepared mixed micellar formulation of paclitaxel comprising Pluronics (either F127 or F68) and low MW heparin all trans retinoic acid (LHR). The latter conjugate was produced by covalently bonding all trans retinoic acid amide to low MW heparin via amide formation. Further mixed micellar formulation was prepared by probe-type sonication and dialysis. An increase in Pluronic:LHR ratio led to a decrease in paclitaxel drug loading. The authors determined 1:4 Pluronic:LHR ratio to be optimal for entrapment efficiency, size, and drug loading. The optimized mixed micellar formulations were stable for a 3-month storage period. The in vitro release was tested in simulated gastric- and intestinal fluids. The mixed micelles released less than 30% of its load during the initial 2 h and a total of 75% 85% load was released in the medium. As heparin underwent desulfation in acidic media, the release rate was increased upon decreasing pH. In vitro cytotoxicity was evaluated by means of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test in MCF-7 cell line. Both tested nanomicellar formulations showed similar cytotoxic effect. And in situ single pass perfusion study was performed in rats, in order to investigate

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absorption of paclitaxel in duodenum and jejunum. A Pgp-mediated efflux was examined by using a Pgp inhibitor, verapamil. Compared to commercial formulation, Taxol, mixed micellar formulations demonstrated enhanced permeability in both small intestine parts. The effect of Pluronics on Pgp was similar to the inhibitory effect of verapamil. Pharmacokinetic study following single oral dose of either Taxol or mixed micellar formulations were performed in rats. The study demonstrated an increase in AUC, cmax and mean residence time (MRT) for mixed micellar formulations. The F68-based formulation was more efficient compared to F127-based system. Bernabeau et al. (2016) solubilized paclitaxel in mixed micellar system composed of TPGS and Soluplus. Soluplus is a new amphiphilic copolymer, the polyvinyl caprolactam polyvinyl acetate polyethylene glycol. It is capable of forming micelles with very low CMC (0.76 3 1023%). The authors compared formulations based on paclitaxel solubilized in mixed micellar system with ones based on single surfactants. Firstly, the CMC value was determined by surface tension method. The experimental CMC value for mixed micellar system was lower than theoretically calculated CMC, presenting a negative deviation from the ideal behavior. The latter indicates a favorable mixing process, probably due to a decrease of hydrophobic interactions among Soluplus chains upon addition of TPGS. As the Soluplus content in mixed micellar system increased, the CP decreased, as the system became more hydrophilic. The micelles composed of pure Soluplus displayed the highest solubilizing capacity for paclitaxel, overall paclitaxel solubility increased at least two orders of magnitude for all evaluated systems. The in vitro release rate showed that simple systems composed of one surfactant released more paclitaxel within 72 h, compared to mixed micellar formulation. The latter showed a sustained release with only 13% of loaded drug released during 24 h. In vitro cytotoxicity studies were performed using three different cancer cell lines and water soluble tetrazolium (WST) assay for viability assessment. Following cell lines were employed: ovarian cancer cells SKOV-3, breast cancer cells MCF-7 and triple negative breast cancer cells MDA-MB-231. While MCF-7 is an estrogen receptor positive tumor, MDA-MB-231 does not express either progesterone, estrogen or human epidermal growth factor type 2 receptors. Mixed micelles could significantly improve the cytotoxic effect of paclitaxel in all three cell lines with different biological behavior. Chan (2016) employed novel organocatalytic drug loading approach involving a chemical conjugation of an anticancer drug doxorubicin to the micellar core through an acid-labile bond. A degradable polymeric micelle system was based on amphiphilic mPEG-b-polycarbonate block copolymers. A series of diblock copolymers comprised a 5-kDa methoxy PEG block and an 11-repeat unit polycarbonate block bearing imidazole, pyridine, phenol or catechol. Then, four different mixed micellar systems were obtained, i.e., phenol/imidazole, phenol/ pyridine, catechol/imidazole and catechol/pyridine. Micelles were loaded with doxorubicin and compared in respect to drug loading, drug release, biodistribution

9.4 Mixed Micellar Formulation for Antineoplastic Agents

in mice and anticancer efficacy in vivo. Authors suggested that following a Raper-Mason-type pathway, doxorubicin became covalently attached to the polymer during the aza-Michael addition step. As a result up to 20% drug loading was achieved for catechol copolymers. Phenol copolymer-based mixed micelles only bound a fraction of doxorubicin, probably due to hydrophobic interactions, proving that the binding mechanism depended on the abovementioned organic catalysis. The optimal system composed of catechol-PEG and imidazole-PEG polymers showed negligible release in neutral media. However, in pH 5, 40% of the drug was released in 8 h showing the pH-responsive properties of the system (Fig. 9.3). The distribution of the nanocarrier was further envisaged in BALB/c mice bearing subcutaneous 4T1 mouse breast tumors using near infrared fluorescent dye. The fluorescence signal was much stronger in tumors in the groups treated with mixed micelles. Moreover, for the mixed micellar nanovesicles no signal was detected in the heart, suggesting lower cardiotoxicity could be expected compared to free doxorubicin treatment. The antineoplastic activity was significantly higher in mixed micelle formulation group compared to group treated with free doxorubicin. Moreover the body weight loss was significantly higher for mixed micelles group. Tumor and heart sections of mice treated with different preparations were

pH 7.4 Urea/acid mixed micelles pH 7.4 Catechol/imidazole mixed micelles pH 5 Catechol/imidazole mixed micelles

100 80

Cumulative release of DOX (%)

Cumulative release of DOX (%)

120

100 80 60 40 20 0

0

20

60 40 Time (h)

80

100

60 40 20 0

0

1

2

3

4 5 Time (h)

6

7

8

9

FIGURE 9.3 In vitro drug release profiles of doxorubicin from catechol/imidazole mixed micelles (covalent bond) at pH 5 and 7.4 and urea- and acid-functionalized mixed micelles (noncovalent interaction). Inset: Release profile for extended period of time. Reprinted with permission from Chan J.M., Tan J.P., Engler A.C., Ke X., Gao S., Yang C., et al., Organocatalytic anticancer drug loading of degradable polymeric mixed micelles via a biomimetic mechanism, Macromolecules 49 (6), 2016, 2013 2021. Copyright 2014 American Chemical Society.

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stained for apoptotic bodies using terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL). For mixed micellar formulation-treated group a high number of apoptotic bodies was detected in tumor and low number in heart. An opposite was observed for group treated with doxorubicin dissolved in saline. An interesting thermosensitive gel system was developed by Sheu et al. (2016). They encapsulated docetaxel in mixed micelles composed of Pluronic F127 and Pluronic L121. Furthermore, mixed micellar formulation was incorporated into 13.5% F127 thermo responsive gel together with doxorubicin. A sustained release was achieved for both drugs. Observation of mixed micellar formulation by TEM imaging revealed that micelles were spherical, nonaggregated with a mean diameter of around 200 nm. Tumor inhibitory rate reached 92.4% and 87.9%, following intratumoral and peritumoral administration in rats, respectively. The antineoplastic efficacy was higher, compared to intravenous injection of free doxorubicin, docetaxel commercial formulation, Tynen, or mixed micellar formulation of docetaxel or subcutaneous injection of docetaxel mixed micellar thermos responsive gel sans doxorubicin. The biodistribution studies showed that following the administration of optimized formulation, the accumulation of doxorubicin in the heart was reduced, compared to plain drug intravenous administration. Secondly, it was possible to decrease systemic levels of docetaxel, and subsequently reduce its toxicity. This can be ascribed to more localized deposition of both agents. Polymeric micelles have previously been used as cancer drug delivery systems in preclinical studies and some formulations based on these systems are currently undergoing clinical trials. SP1049C, a copolymeric micellar formulation of doxorubicin containing Pluronics, has shown prolonged clearance compared to free drug, and higher efficacy in patients suffering from advanced cancer, compared to standard treatment (Valle et al., 2011). It shows activity in anthracycline-resistant tumors and against cancer stem cells (Supratek, 2014). Being a platform drug, it has the potential of reaching blockbuster status. The first indications would include second and third line upper gastrointestinal tract (GI), multidrug resistant lung and breast cancers (Svenson, 2012). It has obtained two orphan drug designations and has a Special Protocol Agreement with the FDA for a Phase III trial in upper GI.

9.5 EXAMPLES OF OTHER MIXED MICELLAR SYSTEMS Rupp et al. (2010) studied solubilization of model hydrophobic drugs: diazepam, tetrazepam, and estradiol in mixed micelles composed of sucrose esters, polyglycerol esters, and hydrogenated phosphatidylcholine (hPC). In preformulation solubility studies, sucrose laurate and sucrose myristate showed susperior solubilizing capacity, compared to Tween 80 and polyglycerol esters. The optimized formulation increased aqueous solubility of diazepam by 35 times (Table 9.2).

9.5 Examples of Other Mixed Micellar Systems

Table 9.2 Examples of Some Mixed Micellar Formulations Authors

Encapsulated Molecule and Mixed Micelle Material

Characteristics of the System

Duan et al. (2015)

Curcumin, Pluronic P123/TPGS (P123:TPGS 5 2.30, w/w) micelles

Horev et al. (2015)

Farnesol, p(DMAEMA)-b-p(DMAEMAco-BMA-co-PRAA)/p(DMAEMA)

El-Dahmy et al. (2014)

Vinpocetine, Pluronic L121/F127 68:32

Zhang et al. (2014)

Stiripentol, monomethoxypoly(ethylene glycol)-b-poly(ε-caprolactone)/sodium oleate micelles Curcumin, MPP:TPGS (4:1)

Blank: 9.50 nm diameter (PDI 5 0.190); ζ-potential 1 0.003 mV Loaded: 10.83 nm diameter (PDI 5 0.114); CMC 0.0163 g/L ζ-Potential—0.069 mV Blank: diameter 21 nm; PDI 5 0.2 Loaded: diameter 60 nm Loaded: 161.5 6 7.39 nm diameter (PDI 5 0.21), ζ-potential 222.42 mV Loaded: 44.2 nm diameter (PDI 5 0.087), ζ-potential 230.3 mV Blank: 31.7 nm diameter (PDI 5 0.255), ζ-potential 10.584 mV, CMC 5.4 mg/L Loaded: 46 m diameter (PDI 5 0.239), ζ-potential 10.613 mV Blank: 22.2 nm diameter (PDI 5 0.309), ζ-potential 25.69 6 0.78 mV, CMC 0.032 mg/mL Loaded: ζ-potential 223.33 6 1.59 mV Blank: 21 6 3 nm diameter, CMC 0.18 mg/mL Loaded: 25 6 2 nm diameter, CMC 0.2 mg/mL

Duan et al. (2016)

Kulthe et al. (2011)

Aceclofenac, P123:L81 (3%:0.5%)

Rupp et al. (2010)

Diazepam, sucrose laurate/hPC (1:1)

BMA, butyl methacrylate; DMAEMA, 2-(dimethylamino)ethyl methacrylate; hPC, hydrogenated phosphatidylcholine; mPP, methoxy poly(ethylene glycol)-poly(lactide); PRAA, 2-propylacrylic acid; TPGS, D-α-tocopheryl polyethylene glycol 1000 succinate.

Zhang et al. (2014) explored the potential of mixed micelles to protect an anticonvulsant drug, stiripentol (STP). They conjugated mPEG and ε-caprolactone via ring-opening polymerization. The obtained diblock copolymer, mPEG-PCl had MW of 10,388 Da and polydispersity index of 1.265. The mixed micellar formulation was obtained by solvent diffusion method, mixing 0.1 g STP with 0.2 g sodium oleate and 0.6 g mPEG-PCl. The DSC thermogram indicated the disappearance of endothermic STP peak upon encapsulation in mixed micelles. The absence

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of STP melting point demonstrated the monomolecular or amorphous state of the encapsulated STP. STP confined within the mixed micellar system avoided degradation in simulated gastric fluid and exhibited sustained release behavior; only around a quarter of initial concentration released during 3 days. The formulation stability was satisfactory, following 3-month investigation. The formulation showed enhanced oral bioavailability following administration in rats. It exhibited 50% higher AUC and doubled t1/2 compared to conventional formulation, Diacomit. Horev et al. (2015) prepared polymeric nanomicelles using pH-responsive diblock copolymers composed of 2-(dimethylamino)ethyl methacrylate (DMAEMA), butyl methacrylate (BMA), and 2-propylacrylic acid (PRAA) (p(DMAEMA)-b-p(DMAEMA-co-BMA-co-PRAA)). While the latter polymer formed pH-responsive micelle core, the DMAEMA was found in micellar coronas. pH-activated nanoparticles are composed of polymers that release the drug following polymer degradation in the acidic dental biofilm. The pH of the oral biofilm matrix comprising extracellular polymeric substances is reported to be around 4.5 5.0 (Horev et al., 2015). The disruption of nanoparticles ensures a selective localized drug release at the biofilm as well as within acidic pH niches, where acidophilic, cariogenic pathogens grow. In this study, protonation of DMAEMA and PRAA in the acidic pH caused the destabilization of nanomicelles and subsequent release of the active agent, farnesol. The monodisperse spherical micelles were found to swell following the addition of farnesol, due to hydrophobic interactions between farnesol and hydrophobic polymer within the micellar core and subsequent formation of farnesol phase within the core. Loading efficiency exceeded 90% throughout the examined nanomicelle compositions, increasing the farnesol solubility to 440 times the level of its solubility in plain water. The protonated amine residues of polymeric micellar corona interacted electrostatically with OH2 and PO432 groups of hydroxyapatite (HA) or alternatively via PO432 bridges with calcium ions exposed on the HA surface. The surface charge-dependent mechanism of binding to salivacoated HA and glucan-coated HA was shown by comparing the absorption rate of micelles of different composition exhibiting distinct zeta potentials. The fact that neutral nanomicelles also exhibited some degree of binding showed alternative mechanisms, such as H-bonding and hydrophobic interactions, and may, to some extent, participate in nanomicelle-HA binding. Nanomicelles exhibited pH-triggered drug release, because the farnesol release rate was twice as fast at pH 4.5, compared to pH 7.2. 75% of farnesol released within 12 h at pH 4.5. Farnesol-loaded micelles exhibited over 2-log reduction of planktonic Streptococcus mutans (S. mutans) viability and were also effective against S. mutans biofilm. Finally, the formulation showed excellent cariostatic effect in S. mutans-infected rodents. Duan et al. (2015) encapsulated curcumin in Pluronic P123/TPGS mixed micelles. In order to improve drug loading and encapsulation efficiency, central composite design-response surface methodology was applied. The following factors

9.5 Examples of Other Mixed Micellar Systems

were included: the mass ratio between surfactants, curcumin dose, hydration method, volume of hydration medium, as well as temperature. Further curcumin-loaded micelles were incorporated into gellan gum gel in order to prolong ocular retention time. The entrapment efficiency was proportional to P123/TPGS ratio, owing to the better hydrophobicity of Pluronic P123. The in situ forming gels, containing curcumin-loaded P123/TPGS micelles, exhibited sustained curcumin release. Mixed micelles did not influence the viscosity of gellan gum in situ forming gels. They also proved to be biocompatible in the irritation tests performed on excised rabbit cornea and irises. Permeation through rabbit corneal tissue was enhanced as nonionic surfactants promoted nanovesicle accumulation in the epithelium and their subsequent permeation across stroma. The authors suggested that the loaded micelles passed through the pores between tight junctions in superficial layer of the corneal epithelium. Alternatively, hydrophilic micellar shells could enable intracellular particle passage through suprabasal and basal layer. El-Dahmy et al. (2014) aimed to obtain a vinpocetine sustained release formulation for intravenous administration. They tested Pluronics P123, F127 and L121 as mixed micelle-forming components and evaluated their influence on formulation properties by using Design-Expert software. The size of the analyzed nanomicelles range from 10.7 to 315.4 nm, PDI was between 0.21 and 0.69, the ζ-potential 230.3 mV was between 218.3 and 235.4 mV and the entrapment efficiency differed considerably, from 7% up to 94%. The optimized formulation exhibited prolonged release in vitro, compared to reference marketed product. The in vivo pharmacokinetic parameters of the experimental and marketed formulations were similar, in respect to AUC. However, the Pluronic mixed micellar system exhibited sustained release with t1/2 increased by 40% and almost doubled MRT. Duan et al. (2016) developed a curcumin formulation intended for oral administration. The mixed micellar system composed of methoxy poly(ethylene glycol)poly(lactide) (mPP) and TPGS was obtained by thin film diffusion method. Single factor experiments were carried out to determine the influence of mixed micelle components on entrapment efficiency, the amount of precipitated drug (PD%), and size. Based on the obtained data, multivariate nonlinear regressions were performed and response surface plots were constructed. It was found that PD% decreased while the mass ratio of MPP to TPGS increased. The higher hydrophobic character of MPP made micelles more dense and stabilized the encapsulated curcumin. However, TPGS also played important role in micelle stabilization, as a hydrophilic micellar corona composed of PEG chains of TPGS prevented micellar aggregation. A proper balancing between both surfactant contents led to choosing an optimal MPP:TPGS mass ratio. The final solubility (1.2 mg/L) significantly exceeded curcumin solubility in plain water. Mixed micellar systems resisted dilution (up to 500 times) in both simulated gastric- and intestinal fluids. However, B21% of loaded drug was lost following incubation with simulated gastric fluid at 1000 3 dilution. A sustained release of curcumin was observed in vitro. Only 4% of curcumin was released in the first 2 h.

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Intestinal absorption was determined in vivo through perfusion experiments in rats; four intestinal segments were analyzed separately. Following oral administration of mixed micellar formulation, curcumin AUC was increased 103 compared to curcumin suspension. The slightly positive charge enabled carrier interaction with negatively charged mucin and thus mucoadhesive properties. The small particle size led to particle diffusion across mucus and subsequently across intestinal epithelium. It was also suggested that TPGS could act as permeation enhancer, due to the inhibition of Pgp efflux. Kulthe et al. (2011) prepared a mixed micellar formulation of aceclofenac by using hydrophobic Pluronic L81 and slightly amphiphilic P123. Although L81 had high potential for solubilization of aceclofenac, it produced large, unstable lamellar structures in aqueous medium. The addition of P123 stabilized small micelles and hindered L81 tendency to aggregate and separate out from aqueous environment, as shown by turbidity measurements. The optimized formulation demonstrated good stability during 1 month storage. Micelles were also stable in the presence of bovine serum albumin during 96 h storage. The release rate from mixed micellar formulation exhibited sustained release of aceclofenac reaching around 23% during 4 h and 45% at 12 h.

9.6 CONCLUSIONS Mixed micellar nanocarriers are versatile drug delivery systems developed in order to address some problems associated with use of nanosized micelles composed of only one copolymeric block. Mixed micellar formulations can be better tuned with solubilized drugs of interest, and subsequently higher drug loading can be achieved. Synergistic interactions between micelle-forming blocks yield micelles that are more thermodynamically stable. Furthermore, the mixed micelles systems may be easily optimized with regard to size and surface charge. Finally, pH-sensitive blocks or surfactant capable of hindering Pgp-mediated drug efflux can be introduced. These nanoparticulate drug delivery systems are also capable of encapsulating a wide range of drugs and can encapsulate two active agents. Their fabrication is simple and they are suitable for the two most popular administration routes: intravenous and oral. We therefore anticipate that they will continue to be a popular option for formulating many new and existing API.

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Zhao, Y., Li, Y., Ge, J., Li, N., Li, L.B., 2014. Pluronic-poly (acrylic acid)-cysteine/ Pluronic L121 mixed micelles improve the oral bioavailability of paclitaxel. Drug Dev. Ind. Pharm. 40 (11), 1483 1493.

FURTHER READING Banipal, T.S., Sood, A.K., 2013. Mixed micellar and interfacial interactions of a triblock polymer (EO37PO56EO37) with a series of monomeric and dimeric surfactants. J. Surf. Detergents 16 (6), 881 891. Ding, H., Yu, H., Dong, Y., Tian, R., Huang, G., Boothman, D.A., et al., 2011b. Photoactivation switch from type II to type I reactions by electron-rich micelles for improved photodynamic therapy of cancer cells under hypoxia. J. Control. Release 156, 276 280. Ebrahim Attia, A.B., Ong, Z.Y., Hedrick, J.L., Lee, P.P., Ee, P.L.R., et al., 2011. Mixed micelles self-assembled from block copolymers for drug delivery. Curr. Opin. Colloid Interface Sci. 16 (3), 182 194. Ganta, S., Devalapally, H., Shahiwala, A., Amiji, M., 2008. A review of stimuliresponsive nanocarriers for drug and gene delivery. J. Control. Release 126 (3), 187 204. Lee, H., Soo, L.P., Liu, J., Butler, M., Allen, C., 2007. Polymeric micelles for formulation of anti-cancer drugs. In: Amiji, M.M. (Ed.), Nanotechnology for Cancer Therapy. CRC Press, Boca Raton, USA, pp. 317 356.