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Design and Assembly of pH-Sensitive Lipidic Cubic Phase Matrices for Drug Release Ewa Nazaruk,† Monika Szlęzak,† Ewa Górecka,† Renata Bilewicz,*,† Yazmin M. Osornio,‡ Peter Uebelhart,‡ and Ehud M. Landau*,‡ †

Department of Chemistry, University of Warsaw, Pasteura 1, PL 02-093 Warsaw, Poland Department of Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland

‡

ABSTRACT: Bicontinuous lipidic cubic phases (LCPs) exhibit a combination of material properties that make them highly interesting for various biomaterial applications: they are nontoxic, biodegradable, optically transparent, thermodynamically stable in excess water, and can incorporate active molecules of virtually any polarity. Here we present a molecular system comprising host lipid, water, and designed lipidic additive, which form a structured, pH-sensitive lipidic matrix for hydrophilic as well as hydrophobic drug incorporation and release. The model drug doxorubicin (Dox) was loaded into the LCP. Tunable interactions with the lipidic matrix led to the observed pH-dependent drug release from the phase. The rate of Dox release from the cubic phase at pH 7.4 was low but increased significantly at more acidic pH. A small amount of a tailored diacidic lipid (lipid 1) added to the monoolein LCP modified the release rate of the drug. Phase identity and structural parameters of pure and doped mesophases were characterized by small-angle X-ray scattering (SAXS), and release profiles from the matrix were monitored electrochemically. Analysis of the release kinetics revealed that the total amount of drug released from the LCP matrix is linearly dependent on the square root of time, implying that the release mechanism proceeds according to the Higuchi model.

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highly attractive for various applications.14 These include the crystallization of proteins, specifically membrane proteins,9,13,15 drug delivery,16,17 biosensing, and biofuel cell construction.18−21 Lipids within bicontinuous LCPs form curved bilayers, which are surrounded by two identical, yet nonintersecting aqueous channel systems. The compartmentalization of LCPs can be utilized to introduce either hydrophilic, lipophilic, or amphiphilic additives and specifically drugs.17,22−24 When using LCPs as hosting layers for modifying electrodes with redox enzymes, it is imperative that the amphiphilic enzyme remains associated with the lipid bilayer, while the aqueous channels allow the reaction substrates and products to diffuse freely through the system. Being highly viscous, LCPs can be easily deposited as thin films onto solid supports, and such films remain stable in the presence of excess of water. Hydrophilic drugs will be located in close proximity to the lipid’s polar headgroup or in the aqueous channels, whereas lipophilic drugs will partition into the lipid bilayer and amphiphilic drugs to the interface between the bilayer and the aqueous compartments.25,26 Diffusion of an incorporated drug from LCP depends primarily on the size and polarity of the molecules, and the rate of transport can be tuned e.g. by

INTRODUCTION Among the various mesophase materials that are formed from hydrated lipids, the gel-like lipidic cubic phase (LCP) stands out as the only material that is transparent and highly structured.1 Remarkably, LCPs are stable with any amount of excess water,2 i.e., at physiological milieus, and have therefore been frequently investigated liquid crystalline structures for drug delivery.3 Hydrated lipids exhibit polymorphism, thereby forming various lyotropic liquid crystalline phases of the inverted self-assembled class (type II) with distinct 1D, 2D, and 3D structures such as lamellar, hexagonal, and cubic phases. Most LCPs utilized to date are based on monoacylglycerols, and among those, “1-monoolein” (1-(cis-9-octadecenoyl)-racglycerol, MO) is the most widely used lipid. Recently, phytantriol (PT) and other isoprenoid-type lipids were shown to form LCPs in excess water (Scheme 1A).4 The cis double bond within MO’s hydrocarbon chain renders its ability to form a wedge structure and to self-assemble into curved bilayers, which is required for the formation of cubic phases upon hydration at ambient temperatures. Generally, the lipid’s structure, temperature, pressure, and hydration determine the unit cell size, curvature, and bilayer thickness of a given LCP.5,6 Lipids that form LCPs are typically nontoxic, biocompatible, and biodegradable. Entrapment of guest molecules, including proteins, within the LCP matrix protects them from chemical and physical degradation, thereby facilitating retention of their native conformation and bioactivity7−13 and rendering LCPs © 2014 American Chemical Society

Received: September 25, 2013 Revised: January 9, 2014 Published: January 20, 2014 1383

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Scheme 1. (A) Structures of the Lipids Used; (B) Redox Process of Doxorubicin

as an oral sustained release delivery system for the poorly water-soluble drug cinnarizine, provided that they are not digested rapidly upon administration.34 The stability of LCPs with excess water constitutes the basis for our studies. The present paper presents an investigation of MO- and PT-based LCPs as pH-sensitive drug delivery systems for Dox, en route to developing LCPs as biomaterials for various applications. Dox is currently used to treat a number of cancer types, including ovarian cancer and multiple myeloma, and was shown to reduce the cardiotoxic side effect on the healthy cells when incorporated into liposomes, thereby allowing the drug to remain longer in the bloodstream, so that more of the drug reaches the cancer cells.35

controlling the charge of the lipids’ headgroup. Thus, incorporation of a negatively charged phospholipid results in slower release of positively charged timolol maleate from the cubic phase, and the amount of drug that was released was inversely proportional to amount of charged phospholipid in the system.27 It was shown recently that release of drugs is significantly faster from LCP than from other mesophases when the area between the liquid crystalline phase and the release medium is constant, the relative diffusion rates being V2 > L2 > H2 > I2 where V2 is bicontinuous cubic phase, H2 inverse hexagonal, I2 micellar cubic phase, and L2 inverse micellar phase.28 Upon variation of the area between the self-assembled system and the release medium, other factors were found to play a role in drug release.26 Mezzenga et al. have demonstrated recently the application of a pH-dependent drug delivery system based on a monolinolein/linoleic acid LCP:29 at pH 2, as is present in the stomach, hexagonal phase was formed and drug diffusion was retarded, whereas at pH 7 LCP was formed and release was ca. 4 times faster. Moreover, the size of the aqueous channel can be tuned by use of additives: addition of the detergent octyl glucoside to MO-based LCP was shown to enlarge the aqueous channel.30 Similarly, addition of a sugar ester as a cosurfactant to monolinolein-based LCP resulted in enhancement of water intake.17 At 10% load, a cubic-Pn3̅m to cubic-Im3m phase transition was induced. The hydrophobic drug capsaicin, embedded within LCP, was investigated for transdermalcontrolled release. It was found that the release mechanism proceeds according to an anomalous transport in which Fickian transport and matrix relaxation occur simultaneously.31 Thus, 34% of the capsaicin was released after 108 h, making such systems useful for sustained release. A cubic phase nanoparticle delivery system was shown to improve the oral bioavailability of poorly water-soluble agents. An in vivo pharmacokinetic study showed that the oral bioavailability of paclitaxel-loaded cubic phase nanoparticles (13.16%) was 2.1 times that of Taxol (the commercial formulation of paclitaxel, 6.39%).32 Release of the lipophilic drugs griseofulvin, rifampicin, diazepam, and propofol from cubosomes was found to reach a plateau value within 20 min.33 It was found that lipids that form liquid crystalline structures in excess water, such as LCPs, may have application

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MATERIALS AND METHODS

Monoolein (1-oleoyl-rac-glycerol) (MO), MES (2-(N-morpholino)ethanesulfonic acid), HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid), TRIZMA (tris(hydroxymethyl)aminomethane hydrochloride), and Doxorubicin (Dox) were purchased from Sigma and were used as received. Phytantriol (PT) was obtained from DSM as a gift. Lipid 1 (Scheme 1A) was synthesized according to ref 36. McIlvaine buffer was prepared by mixing 0.1 M citric acid and 0.2 M disodium phosphate (both from POCh (Polish Chemicals Co.)). All solutions were prepared using Milli-Q water (18.2 MΩ cm−1; Millipore, Bedford, MA). Preparation of LCPs: Appropriate amount of water was added to molten MO or PT in a small glass vial. The ratio of components was chosen on the basis of the phase diagrams for the MO/water37 or PT/ water systems.4 LCPs doped with Dox were prepared by first mixing Dox with MO or PT to obtain a homogeneous system and subsequently adding water. The ratios for the MO/H2O/Dox and PT/H2O/Dox were 63/ 36/1 and 72/27/1% (w/w), respectively. In the case of LCPs containing lipid 1 as additive, the lipids were initially melted, mixed with Dox, and subsequently mixed with water. To obtain homogeneous, transparent and viscous LCPs, samples were centrifuged for ca. 1 h in a MPW 56 centrifuge. Samples were stored in tightly closed vials at room temperature in the dark. Phase identity and structural parameters of the lipidic samples were determined by X-ray scattering using a Bruker GADDS system working with Cu Kα radiation and a Bruker Nanostar system working with Cu Kα radiation equipped with a Vantec 2000 area detector. Polarized microscopy was conducted with a Nikon Eclipse E400 1384

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microscope equipped with a LINKAM THMS 600 heating/cooling stage. Electrochemical measurements were performed using a CHI bipotentiostat in a three-electrode arrangement with a SCE reference electrode and a platinum foil as the counter electrode. The working electrode was glassy carbon (GCE) modified with the MO or PT cubic phase film. Prior to the measurements, the electrode was polished on alumina (0.1 and 0.05 μm) with a polishing cloth. The electrodes were then rinsed with water in the ultrasonic bath and left to dry. For each type of cubic phase triplicate experiments were performed.

the intensity ratios of the X-ray signals for the sample doped with lipid 1 and the nondoped one are similar (Figure 1A), it can be concluded that the electron density, and thus the molecular distribution, is not significantly altered by incorporating lipid 1 into the matrix. X-ray data collected for the nondoped PT/H2O system (containing 27% w/w water) show the formation of a cubicPn3̅m structure at 25 °C, with lattice parameter a of 64.5 Å. The cubic-Pn3̅m phase transforms into the reverse hexagonal phase HII with lattice parameter a of 45.8 Å at 45 °C. At 63 °C, the HII phase transforms into the isophase. When doped with 1% (w/w) of Dox, the phase behavior is altered: A cubic-Ia3d phase with lattice parameter a of 100.5 Å is formed at room temperature. Above 29 °C, the cubic-Ia3d undergoes a phase transformations to the cubic-Pn3̅m phase, which in turn transforms into an HII phase above 45 °C and to the isophase at 62 °C. At a level of 1% (w/w) Dox the unit cell parameters for both the Pn3̅m and HII phases are comparable to the nondoped systems. When the Dox content is increased to 6% (w/w), the cubic-Ia3d phase exists in the temperature range from room temperature to 38 °C. Thus, we conclude that addition of a small amount of Dox (∼1% w/w) to both MO- and PT-based lipidic cubic phase systems does not alter significantly the properties of the system; both systems preserve the cubic structures at room temperature. Moreover, incorporating the diacidic lipid 1 into the MO/H2O/Dox system results in only a small increase of the crystallographic unit cell parameter at temperatures above 45 °C (Table 1 and Figure 1). Electrochemical Behavior of Doxorubicin Incorporated in the Cubic Phase. Because of the presence of the quinone and hydroquinone group, Dox is electroactive and its behavior in LCP can be monitored electrochemically (Scheme 1B). At pH 4.5 a pair of reversible peaks can be observed at ca. −0.5 V, which are related to the reduction and oxidation of quinone redox group undergoing 2e/2H+ processes.40 The electrode processes of Dox in aqueous solution were investigated at room temperature using glassy carbon electrodes modified with nondoped MO-based LCPs. The electrode was immersed in McIlvaine buffer solution at pH 4.5 containing Dox at 10−5 M concentration. Full saturation of the cubic phase with Dox was achieved after 500 min (Figure 2A). The cyclic voltammogram recorded for the electrode modified with the LCP and saturated with Dox is presented in Figure 2B. On the basis of the dependence of peak current on the square root of scan rate, it was found that the process is diffusion controlled. Dox was incorporated into the MO matrix at concentrations of 0.3, 0.6, and 1.1% (w/w). Peak current increases with amount of Dox in the cubic phase, and at 1.1% (w/w) the current reaches a plateau. This Dox concentration was therefore applied in all subsequent experiments. Significantly, current was not dependent on the mass of cubic phase placed on the electrode surface up to ca. 150 mg cm−2 (Figure 2C). At higher loads the electrode was blocked and signal was irreversible. A Dox release profile was evaluated with electrochemical methods (DPV and CV). The fraction of Dox released as a function of time for all concentrations was of the same order of magnitude, leading to the conclusion that drug release under these conditions is independent of the initial drug loading at the investigated Dox concentration range. Location of the drug is an important parameter affecting the release rate. In the case of a hydrophobic drug that can be embedded in the lipid bilayer

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RESULTS AND DISCUSSION The cubic phase is unique among all hydrated lipidic mesophases, as it is highly viscous and transparent, due to the lack of birefringence. LCPs can therefore be detected by visual inspection using direct and cross-polarized microscopy. To confirm and evaluate the effect of addition of Dox and of the amphiphilic diacidic compound lipid 1 on the cubic phase type and structure, X-ray scattering and cross-polarized microscopy were conducted. X-ray Data for Doped and Nondoped Samples. X-ray scattering measurements were conducted to identify the type and structural parameters of the liquid crystalline phases. In the wide angle regime (WAXS) all the mesophases exhibited the diffused signal positioned at the angle that corresponds to 4.5 Å. This value is characteristic for phases with liquid-like order of the alkyl chains. In the small-angle regime (SAXS) sharp Bragg reflections characteristic of the long-range positional order were detected. The SAXS data are shown in Table 1. The phase identity and structural parameters for reference MO/ H2O and PT/H2O systems are in accord with published values.4,38,39 Table 1. Composition, Phase Identity and Crystallographic Unit Cell Parameters for the Observed Liquid Crystalline Phases Investigated phase composition (% w/w) PT/H2O (73/27) MO/H2O (64/36) PT/H2O/Dox (72/27/1)

MO/H2O/Dox (63/36/1)

MO/H2O/Dox/lipid 1 (61/36/1/2)

temperature (°C) and phase symmetry 25; 45; 25; 60; 25; 29; 45; 25; 45; 50; 25; 45; 50;

Pn3̅m HII Ia3d Pn3̅m Ia3d Pn3̅m HII Pn3̅m Pn3m ̅ Pn3̅m Pn3̅m Pn3̅m Pn3m ̅

unit cell [Å] a a a a a a a a a a a a a

= = = = = = = = = = = = =

64.5 45.8 145.0 68.0 100.5 64.0 46.5 95.9 90.2 85.0 94.0 91.5 88.8

Sample MO/H2O/Dox (62.9/36/1.1% w/w) exhibited a cubic-Pn3̅m phase in the temperature range studied, 25−90 °C, above which a hexagonal phase was observed. As expected, the crystallographic unit cell shows a negative thermal expansion (Figure 1B). Upon addition of 2% (w/w) of the diacidic lipid 1 the Pn3̅m structure is preserved (Figure 1A), but the lattice parameter increases (by 1−2 Å) in a temperature range above to 45 °C (Figure 1B). In both samples below 45 °C the thermal expansion of the lattice parameters becomes smaller, which may be due to a glass transition in which the Pn3̅m structure is preserved but the molecular motions are frozen. However, since 1385

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Figure 1. (A) 2D X-ray scattering pattern of the cubic-Pn3̅m phase observed for the MO/H2O/Dox/Lipid 1 system and X-ray signals vs 2θ obtained by azimuthal integration of the X-ray intensities from 2D X-ray scattering patterns of the MO/H2O/Dox/Lipid 1 and MO/H2O/Dox systems. (B) Temperature dependence of the lattice parameter a of the cubic Pn3̅m phase for the MO/H2O/Dox/Lipid 1 and MO/H2O/Dox systems.

Figure 2. (A) Saturation of the MO-based cubic phase with Dox (10−5 M). (B) Cyclic voltammogram recorded for the electrode modified with cubic phase and saturated with Dox. (C) Dependence of measured peak current on the LCP mass per area of the electrode.

Figure 3. (A) Cyclic voltammogram on GC electrode modified with MO-based (black trace) and PT-based (red trace) LCPs doped with 1.1% (w/ w) Dox. The y-axis depicts the peak current per mole of Dox deposited on the electrode. (B, C) Dependence of the peak current on the square root of scan rate for the MO- and PT-based cubic phases, respectively.

compartment of the cubic phase matrix, the release depends on the partitioning between the lipidic and hydrophilic domains. Electrochemical methods were also employed to compare the behavior of Dox in MO- and PT-based cubic phases. Dividing the peak current by the amount of Dox deposited on the electrode yields a normalized Dox diffusion rate, which was found to be lower in the PT-based LCP than in the MO-based phase under identical conditions (Figure 3). This may be due to the different cubic phase type, which at 25 °C is Pn3̅m and Ia3d for the MO and PT systems, respectively (Table 1), and to the higher viscosity of PT-based LCP. On the basis of dependence of peak current on the square root of scan rate, it was found that in both cases the process was diffusion controlled (Figure 3B,C).

The method for determining the diffusion coefficient in LCPs using electrochemical means was described earlier.41 The diffusion coefficients for Dox at pH 5.8 determined from the voltammetric curves were found to be (4.56 ± 0.31) × 10−9 and (2.25 ± 0.35) × 10−8 cm2 s−1 for PT/H2O/Dox and MO/ H2O/Dox, respectively. Increasing the pH to 7.5 results in significant decrease of diffusion coefficient of Dox embedded in the MO-based LCP, which was found to be (4.36 ± 0.42) × 10−9 cm2 s−1. Doxorubicin Release Profile as a Function of pH. The effect of pH and the affinity of the solubilized drug to lipid bilayers were recently studied.27,42,43 In our investigation the pH dependence of Dox diffusion from the PT- and MO-based LCPs was established by electrochemical methods. DPV on GCE electrode modified with 1.1% (w/w) Dox-doped MO 1386

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plateau. Because at pH 7.5 and 9.0 Dox molecules are predominantly incorporated into the lipid bilayer, penetration into the aqueous channels may become the rate-limiting step. Thus, at pH 7.5 less than 10% of Dox is released after 45 min. Dox release from the PT-based cubic phase was much slower than that from the MO-based cubic phase, as can be seen in Figure 5C: T50 was as large as 5000 min. PT-based LCP can therefore be considered as a potential matrix for slow sustained release of incorporated Dox. Formation of pH-Sensitive Cubic Phase. Up to 10% (w/ w) of lipid 1 can be added to a MO-based LCP without noticeable perturbation of the cubic phase texture, as evidenced by optical microscopy [Osornio, Y. M., personal communication]. Dox-containing MO-based LCP was doped with 2% w/w of the diacidic lipid 1 in order to prepare a cubic phase matrix that is pH-sensitive (Figure 6). The two carboxylate groups in the headgroup region of lipid 1 are exposed to the LCP’s aqueous channel, resulting in charging of the hydrophilic/ hydrophobic interface. As a consequence, Dox release is faster at pH 7.5 than at pH 5.8 (Figure 6B,D), presumably due to electrostatic attraction between the negatively charged headgroup of lipid 1 and the positively charged Dox at the lower pH. In contrast, in the absence of lipid 1 Dox release from the uncharged LCP is faster at pH 5.8 than at pH 7.5. At pH 9 the majority of Dox (87%) is unprotonated and hence located in the lipidic bilayer domain. Charging of the interface by addition of lipid 1 to the cubic phase at this pH results in slower Dox release from the LCP as compared to the release in the absence of lipid 1 up to ca. 260 min. This may be due to slow transfer of Dox from the lipidic to aqueous LCP domains (Figure 6 F). At longer times, an increase in the release rate of Dox in the presence of lipid 1 occurs, whereas in the uncharged LCP release is slowed down, indicating that lipid 1 can control the speed of removal of the drug from the film. This change in the kinetics of release over time makes it impossible to fit the plot of current vs time for this case to any of the mechanisms describing kinetics of drug release from the phase to the solution. At the three pH values investigated Dox potential is shifted to more positive values, and peak current is larger upon doping the LCP with lipid 1, as evidenced by differential pulse voltammetry (Figure 6A,C,E). This may suggest that in the presence of lipid 1 at the hydrophobic−hydrophilic interface of the LCP, a higher fraction of Dox partitions into the aqueous channel domain. Kinetics. An ideal profile of drug release from a prolonged release carrier obeys zero-order kinetics. In the case of LCPs,

LCP at pH 9.0, 7.5, and pH 5.8 is depicted in Figure 4. The potential of maximum peak is shifted toward positive potentials

Figure 4. DPV recorded on GCE electrode modified with Dox-doped MO LCP at pH 9.0, 7.5, and 5.8.

as the pH of electrolyte is decreased indicating that protons were involved in the electrochemical process. The formal potential of Dox was plotted versus pH values; the slope was −60 mV/pH, as expected for a 2e/2H+ electrode process. Figure 5A depicts the time dependence of the peak current. The initial current at pH 5.8 is ca. 5 times higher than those at pH 7.5 and 9.0, indicating that at the low pH Dox resides mainly in the aqueous channels, where diffusion is faster than in the lipid bilayer domains. Because the pKa value of Dox is 8.2, its charge is strongly pH dependent in the investigated pH range: 99.6% of Dox is protonated at pH 5.8, whereas the fraction of uncharged Dox increases to 16.6% at pH 7.5 and to 87% at pH 9.0. Uncharged Dox is expected to reside mainly in the lipidic domain of the cubic phase, while the charged species partitions predominantly into the aqueous channel compartment. Figure 5B clearly demonstrates a faster release from the cubic phase at pH 5.8 than at pH 7.5 and 9.0. At pH 9.0 Dox exists mainly in the unprotonated state, having higher affinity to the lipidic domain and hence its release is much slower. Such pH dependence of the rate of Dox delivery from the cubic phase may be utilized for controlling drug release into tumor cells, since the pH in cancer cells is lower than that of healthy cells. In buffer solution of pH 5.8, the time required for release of half of the total amount of drug incorporated in the LCP (T50 value) is 45 min. At ca. 100 min the Dox current reaches a

Figure 5. (A) Release profiles of Dox from a MO/H2O/Dox (63/36/1% w/w) cubic phase, in buffers, pH 9.0, 7.4, and 5.8. (B) Release profiles of Dox from a MO/H2O/Dox (63/36/1% w/w) cubic phase, in buffers, pH 9.0, 7.4, and 5.8, plotted as normalized current I/I0 vs time. (C) Release profile of Dox from a PT/H2O/Dox (72/27/1% w/w) cubic phase at pH 5.8, plotted as normalized current (I/I0) vs time. 1387

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Figure 6. Left panel: DPV on GCE electrode coated with Dox-containing MO LCP with and without 2% w/w of the lipid 1 additive at pH 5.8 (A), 7.5 (C), and 9.0 (E). Right panel: release profile of Dox from MO-based LCP with and without 2% w/w of the lipid 1 additive at pH 5.8 (B), 7.5 (D), and 9.0 (F), plotted as normalized current (I/I0) vs time.

to non-Fickian transport, n = 0.89 to case II (relaxational) transport, and n > 0.89 to super case II transport. Kinetic data obtained in this study are shown in Table 2 and are compared with data obtained with the model hydrophilic

diffusion of soluble drugs occurs within the continuous aqueous channels, while lipophilic drugs are predominantly incorporated in the lipid bilayer and thus partitioning into the aqueous channel may become the rate-limiting step. A number of theoretical and empirical drug release models have been described in the literature. Specifically for drug release from cubic phases, Peppas, first-order kinetics, and Higuchi models44 have been used. The Higuchi model describes drug release as a diffusion process based on Fick’s law given by the equation M t /M∞ = kt 0.5

Table 2. Evaluation of the Drug Release Kinetics of Using Peppas, Higuchi, and First-Order Models Peppas PT/H2O/Dox MO/H2O/Dox (pH 5.8) MO/H2O/Dox/lipid 1 (pH 7.5) MO/H2O/Dox/lipid 1 (pH 9.0) MO/H2O/VitK3 MO/H2O/K3[Fe(CN)6]

(1)

where Mt/M∞ is a fraction of drug released at time t and k is the release rate constant. In this model the cumulative drug release is proportional to the square root of time. It describes the rate of drug release from a matrix where the drug loading exceeds the solubility in the matrix medium. The other model used for the characterization of drug release is the first-order (I-order) model described by the equation log(M t /M∞) = kt

a

I-order

n

R2

R2

0.996 0.990 0.9731

0.861 ± 0.04 0.605 ± 0.06 0.705 ± 0.05

0.997 0.993 0.992

0.983 0.987 0.986

−a

−a

0.882

0.980

0.998 0.9938

0.534 ± 0.04 0.512 ± 0.01

0.999 0.999

0.983 0.930

Could not fit to this model.

compounds K3[Fe(CN)6] and vitamin K3. Applying Peppas model, data were plotted as log percentage of drug release versus log time. Values of the correlation coefficient for the obtained release data were all higher than 0.97. The release exponent n was higher than 0.5 in all cases, which indicates non-Fickian anomalous transport in which both diffusion and matrix effects are present. Such complex release of Dox can be attributed to a matrix swelling or dissolution process. Using the Higuchi model, the amount of released compounds was plotted against the square root of time,

(2)

To describe drug release from a polymeric system, the model proposed by Peppas was used: M t /M∞ = kt n

Higuchi

R2

cubic phase composition

(3)

In this model, the value of n characterizes the drug release mechanism. In the case of a cylindrical-shaped matrix, n ≤ 0.45 corresponds to a Fickian diffusion mechanism, 0.45 < n < 0.89 1388

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Figure 7. Kinetics of Dox release from the PT, MO, and lipid 1-doped MO LCP at pH 5.8. Y-axis: the fraction of doxorubicin molecules released to the solution. (A) Higuchi model; (B, C) I-order model.

yielding linear relationships with very high correlation coefficients (R2 = 0.999) for diffusion of the model hydrophilic compounds from LCPs, indicating that release is diffusion controlled. Such data are in agreement with the Peppas model, where the n value was close to 0.5, indicating that the diffusion process is based on Fick’s law. Lower R2 correlation coefficients were obtained for Dox embedded into MO- or PT-based cubic phases, which can be explained by the pH-dependent nature of Dox. At pH 5.8 99.6% of the Dox molecules are protonated and are predominantly located in the LCP’s aqueous channels. When fitted to the Higuchi model, these data result in a lower correlation coefficient of 0.993, suggesting that both diffusion and matrix effect influence the release mechanism. The exponent value n of 0.605 ± 0.06 extracted from Peppas model also suggests non-Fickian transport. At pH 7.5, 16.6% of Dox is unprotonated and partitions into the lipidic domain. The n value obtained for this system (0.705 ± 0.05) and the low correlation coefficient point to deviation from simple diffusive transport. In case of Dox embedded into PT-based cubic phase, the drug release proceeds according to a zero-order mechanism. Release data could be fitted to the I-order model. The plots were close to linear, but the correlation coefficient was poor (Figure 7B). Data obtained for the hydrophilic compounds and Dox embedded into the MO-based cubic phase at pH 5.8 suggest that the release mechanism proceeds according to the Higuchi model, whereas in case where Dox can be incorporated into the lipidic domain non-Fickian transport dominates.

the pH responsive lipid 1 to the LCP, resulting in higher reduction peak currents compared to the corresponding nondoped LCPs at all pH values studied. This may suggest that the carboxylate headgroup of lipid 1 affects the charging of the LCP’s hydrophobic−hydrophilic interface, enabling higher loading of Dox in the aqueous channels, where diffusion is much faster than in the lipidic domains. Kinetics of the drug release is enhanced accordingly. Kinetic data obtained for hydrophilic probes ([Fe(CN)6]3− and vitamin K3) as well as for positively charged Dox at pH 5.8 suggest that release mechanism is in agreement with the Higuchi model. At higher pH values, at which Dox is uncharged and readily incorporated into the lipidic domain, non-Fickian transport dominates. The stability of LCP in excess water, its gel consistence, the ability to incorporate active moleculesincluding membrane proteinsof various sizes and polarities, and to stabilize their native conformation are of great potential en route to developing LCPs as biomaterials for various applications. The current investigation demonstrates that designed lipid additives may be implemented to render LCPs pH active, thereby modulating the partitioning of active molecules between the LCP compartments and influencing their release kinetics. Further SAXS studies will reveal the effect of varying pH on the mesophase structure and should be helpful in understanding the drug release behavior as a function of pH. Additional modifications of the host matrix structure and its phase behavior, as well as the guest active compound, will result in fine-tuning of such systems and improved biomaterials for drug delivery and release.

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CONCLUSIONS MO- and PT-based LCPs were investigated as potential matrices for pH-dependent drug delivery. Dox, used in cancer chemotherapy, is a hydrophobic molecule containing an amine group whose pKa is 8.2. Incorporation of up to 1.1% (w/w) of this model drug into the LCP systems does not significantly affect the liquid crystalline structure at room temperature, as evidenced by SAXS measurements. Further addition of 2% (w/ w) of the diacidic additive lipid 1 to the PT/H2O/Dox system does not affect the LCP symmetry, which remains Pn3̅m. On the basis of the voltammetric reduction peak of Dox, it was found that release kinetics is independent of the initial drug loading. Partitioning of the guest drug molecule between the LCPs lipidic and aqueous domains is crucial for the release rate, which is furthermore pH-dependent. The release rate of loaded Dox was slow at pH 7.5 and increased significantly at pH 5.8. At pH 9.0 Dox is expected to partition preferentially into the lipidic compartment of the LCP, and its release rate is even slower. Dox release was further modulated by the addition of

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (R.B.). *E-mail [email protected] (E.M.L.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grant PSPB 079/2010 to R.B. and E.M.L. through the Swiss Contribution to the enlarged European Union.

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