Article pubs.acs.org/Langmuir
Remote Loading of Aloe Emodin in Gemini-Based Cationic Liposomes Chiara Giuliani,†,‡ Barbara Altieri,§ Cecilia Bombelli,*,‡ Luciano Galantini,† Giovanna Mancini,*,∥ and Annarita Stringaro⊥ †
Dipartimento di Chimica and ‡CNR-IMC sez. Meccanismi di Reazione c/o Dipartimento di Chimica, Università di Roma “La Sapienza”, P. le A. Moro 5, 00185 Roma, Italy § Dipartimento di Scienze Fisiche e Chimiche, Università degli Studi dell’Aquila, Via Giovanni Falcone 25, 67100 L’Aquila, Italy ∥ CNR-IMC Istituto di Metodologie Chimiche, Area della Ricerca di Roma 1, Via Salaria Km 29, 300 00015 Monterotondo, Italy ⊥ Dipartimento di Tecnologie e Salute, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Roma, Italy S Supporting Information *
ABSTRACT: Anthraquinone compound aloe-emodin (AE) has shown antineoplastic, antibacterial, antiviral, and antiinflammatory properties and scavenging activity on free radicals. Because of these therapeutic features, AE has been attracting increasing interest and could be applied in the curing of many diseases. However, until now the physicochemical features of this compound have not been fully investigated; furthermore, its wide application might be hindered by its scarce solubility in aqueous media (∼19 μM). The inclusion of AE in nanocarriers, such as cationic liposomes, could allow its delivery effectively and selectively to target sites, reducing side effects in the remaining tissues. In this work, the weak acid nature of AE, because of its two phenolic functions, was exploited to load it remotely in the internal aqueous phase of liposomes in response to a difference in pH between the inside and outside of the liposomes, pHin > pHout. The inclusion of AE in gemini-based cationic liposomes by the acetate gradient method was obtained at high AE/lipid ratios (up to 1:30).
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INTRODUCTION Aloe-emodin (1,8-dihydroxy-3-(hydroxymethyl)-9,10-anthracenedione, Chart 1), AE, is an anthraquinone compound
solubility in water, (ii) controlling its pharmacokinetics and biodistribution, and (iii) increasing its accumulation in target tissues while avoiding or reducing its accumulation in healthy tissues, thus reducing its toxicity and side effects. However, to the best of our knowledge, only two reports deal with the inclusion of the whole extract of aloe vera10,11 in liposomes, and only one report deals with the inclusion of AE in the lipid bilayer of cationic liposomes.12 Indeed, the acidic properties of AE could be exploited to load it into the liposome aqueous compartment by a “remote (active) loading” technique13 that involves a higher entrapment efficacy compared to inclusion in the lipid bilayer. In fact, compounds with acidic/basic properties can be concentrated into the liposome interior space in response to specific pH transmembrane gradients (pHin > pHext/pHin < pHext). This method exploits the different permeability of the lipid bilayer with respect to the charged and the neutral form of many small lipophilic molecules containing titratable groups because the lipid bilayer is much more permeable to neutral molecules than to charged ones. The first successful example of the application of this method concerns
Chart 1. Molecular Structure of Cationic Gemini Surfactant (2S,3S)-2,3-Dimethoxy-1,4-bis(N-hexadecyl-N,Ndimethylammonium) Butane Bromide (1) and Aloe Emodin, AE
contained in the exudate from the inner epidermal layers of aloe leaves. This anthracene derivative is a polyphenol that has been reported to possess many therapeutic properties, such as antineoplastic,1−5 antibacterial,6 anti-inflammatory,7 and scavenging activity on free radicals8 acting through several cellular mechanisms and pathways.9 Because of its properties, AE is attracting increasing interest for its application in the treatment of many diseases. The inclusion of AE in a nanocarrier could enlarge and improve its application by (i) increasing its © 2014 American Chemical Society
Received: September 26, 2014 Revised: November 9, 2014 Published: December 12, 2014 76
DOI: 10.1021/la5038074 Langmuir 2015, 31, 76−82
Article
Langmuir
Influence of Calcium on AE pKa. A concentrated isopropanol solution of AE (200 μL, 480 mM) was diluted (38 μM) in 5.0 mM aqueous CaCl2 at pH 8.2. The calcium concentration was increased by the addition of known volumes of 0.50 M aqueous CaCl2. Absorbance was measured after each addition between 200 and 600 nm. Fluorescence Measurements. The solutions of AE used for UV−vis measurements were diluted in order to obtain samples with optical densities lower than 0.05 to minimize inner filter effects. Fluorescence emission spectra were recorded at an excitation wavelength of 432 nm, collecting the emission signal between 452 and 700 nm. Preparation of Unilamellar Vesicles. Lipid films were prepared on the inside wall of a round-bottomed flask by the evaporation of CHCl3 solutions containing the proper amount of lipids (DMPC, DOPC, POPC, DPPC, cholesterol, and the gemini surfactant). The obtained films were stored overnight under reduced pressure (0.4 mbar) and then hydrated using 170 mM calcium acetate solution at pH 6. Vigorous shaking of lipids, using a vortex mixer above Tm, gave the formation of a heterogeneous population of multilamellar vesicles, MLVs. Obtained MLVs were first freeze−thawed six times, from liquid nitrogen to a temperature 10 °C above Tm of the liposome formulation, and then extruded (10 times) through a 100 nm polycarbonate membrane (Whatman Nucleopore). Extrusions were carried out above Tm using a 2.5 mL extruder (Lipex Biomembranes, Vancouver, Canada). The transmembrane pH gradient was generated by a transmembrane difference in calcium acetate concentration. The acetate salt of the bulk was removed by gel-exclusion chromatography on Sephadex G-50 minicolumns preequilibrated with 170 mM sodium sulfate using the dry filtration protocol.28 All of the formulations were characterized with respect to the vesicle size distribution and zeta and surface potentials before the generation of the pH gradient. Measurement of the Internal pH of Liposomes. The pH of the internal aqueous compartment of lipid vesicles, after the generation of the calcium acetate gradient, was measured by exploiting the fluorescence properties of the membrane-impermeant, pH-sensitive fluorescent molecule, pyranine. Liposome suspensions were prepared as described above. Dry lipid films were hydrated with a solution containing 0.5 mM pyranine in 170 mM calcium acetate at pH 6 and a total lipids concentration of 12 mM. A transmembrane difference in calcium acetate concentration was generated by gel filtration as described above. In this case, the filtration on Sephadex G-50 minicolumns also removed unentrapped pyranine from the bulk. After filtration, the fluorescence emission intensity at 507 nm was measured at excitation wavelengths of 460 and 415 nm. The lipid concentration in the cuvette was 0.03% (w/v), and the DPX quencher was added to the suspension at a 2 mM final concentration to quench the fluorescence of any residual unentrapped pyranine. The internal pH was calculated from the ratio of the fluorescence intensities, R = F460/ F415, using eq 1, as described previously,17
the remote loading of the anticancer anthracycline doxorubicin (a weak base) in pegylated liposomes, a formulation that has been on the market since 1995 (Doxil).14 Other examples concern the loading of alkaloid Oxymatrine15 and some compounds bearing carboxylic functions, such as diclofenac,16 carboxyfluorescein, and nalidixic acid.17 To the best of our knowledge, only one European patent is related to the remote loading of phenolic compounds.18 Herein we report on the loading of AE into gemini-based cationic liposomes by a remote loading approach in which a calcium acetate gradient creates a transmembrane pH imbalance.17 A number of different cationic liposomes formulated with a natural phospholipid within 1,2-dimyristoyl-sn-glycero-3-phospholcholine (DMPC), 1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine, (POPC), gemini surfactant (2S,3S)-2,3dimethoxy-1,4-bis(N-hexadecyl-N,N-dimethylammonium) butane bromide (1) (Chart 1), and cholesterol were explored for the inclusion of AE. In fact, it is known that the positive charge on the liposome surface facilitates their interaction with the cell membrane19,20 and attributes to the nanocarriers an intrinsic specificity toward certain tissues, such as lung, liver, and the endothelium of tumor vessels.21 In particular, cationic liposomes formulated with gemini 1 were shown to be more efficient in the delivery of a photosensitizer22−25 and a plasmid DNA26 to cells with respect to both neutral liposomes and a commercial formulation/transfection kit. Moreover, they were shown to be characterized by low toxicity. All liposome formulations explored for the encapsulation of AE were characterized with respect to their vesicle size distribution, zeta and surface potentials, internal pH, and entrapment efficacy, E.E.
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EXPERIMENTAL SECTION
Materials and Methods. Phospholipids DMPC, DOPC, DPPC, and POPC were purchased from Avanti Polar Lipids (Alabaster, AL) and used without further purification (purity >99%). (2S,3S)-2,3Dimethoxy-1,4-bis(N-hexadecyl-N,N-dimethylammonium) butane bromide was prepared and purified as previously described.27 Aloeemodin, 8-hydroxypyrene-1,3,6-trisulfonic acid (pyranine), and 4heptadecyl-7-hydroxycoumarin (C17-HC) with purities of greater than 95, 97, and 98%, respectively, were purchased from Sigma-Aldrich and used without further purification. p-Xylene-bis-pyridinium bromide (DPX) was purchased from Invitrogen (Oregon, USA). PBS (0.01 M phosphate buffer, 0.0027 M KCl, 0.137 M NaCl, pH 7.4 at 25 °C), calcium acetate, Sephadex G-50, and sodium sulfate from SigmaAldrich were also used. Absorption and fluorescence emission spectra were recorded in a quartz cuvette of 1.0 cm path length by using a Cary 300 UV−vis double-beam spectrophotometer and a Fluoromax-4 Horiba-Jobin Yvon spectrofluorometer, respectively. Characterization of AE. UV−Vis Measurements. Weighed amounts of AE were dissolved in known volumes of PBS NaOH 1 M, DMSO, chloroform, isopropanol and isopropanol/PBS 1:1 to obtain a 19 μM concentration of AE, and the absorbance was measured between 200 and 600 nm. Determination of AE pKa. A 500 mM AE stock solution in isopropanol was diluted to 40 mM in PBS at pH 7.4. The solution was first acidified to pH 2.3 by the addition of aqueous 1 M HCl, and then the extent of ionization of AE phenolic groups was monitored spectroscopically upon addition of proper amounts of aqueous 0.1 M NaOH. The 2.3−12.6 pH range was explored, and the absorbance of the solutions was scanned in the 350−650 nm range. Data were analyzed by plotting the absorbance at 504 nm versus pH.
⎛ R − Ra ⎞ pH = pK a + log⎜ ⎟ ⎝ Rh − R ⎠
(1)
where pKa = 7.268 ± 0.003 is the apparent pKa of the fluorescent probe and Ra = 0.0122 ± 0.0007 and Rb = 2.515 ± 0.006 are the F460/ F415 values of the protonated form and the unprotonated form of the probe, respectively. Liposome Size Determination by Dynamic Light Scattering Measurements. The size and the size distribution of the lipid aggregates (1.2 mM total lipid concentration, 1.7 mM calcium acetate solution, pH 6) were evaluated by dynamic light scattering (DLS) measurements. A Malvern NanoZetasizer apparatus equipped with a 4 mW HeNe laser source (632.8 nm) was used. In this apparatus, the light scattered by the sample, placed in a thermostated cell holder, is collected at an angle of 173°. To obtain the size distribution, we analyzed the measured autocorrelation functions by means of the CONTIN algorithm. Decay times were used to determine the distribution of the diffusion coefficients D of the particles, which in turn were converted in a distribution of apparent hydrodynamic 77
DOI: 10.1021/la5038074 Langmuir 2015, 31, 76−82
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Langmuir diameters dH using the Stokes−Einstein relation, dH = kBT/3πηD where kBT is the thermal energy and η is the solvent viscosity. The values of the diameters reported herein correspond to the average values over several measurements on three independent liposome samples and were obtained from intensity-weighted distributions. Zeta Potential Measurements. DMPC, DPPC, DOPC, POPC liposomes, mixed liposomes containing a phospholipid (DMPC, DPPC, DOPC, or POPC), cationic surfactant 1, and cholesterol in a 1.6:0.4:1 molar ratio were prepared in 17 mM calcium acetate at pH 6 at a total lipid concentration of 1.2 mM. The measurements of the electrophoretic mobility to determine the zeta potential were carried out by means of the laser Doppler electrophoresis technique using the same apparatus used for DLS measurements. Zeta potential values were obtained by means of the electrophoretic mobility of liposomes in the electrolyte solution upon the application of an electric field, as described by the Henry equation (eq 2), which expresses the velocity of a particle in a unitary electric field
UE =
2εζf (κa) 3η
prepared by hydration with 170 mM calcium acetate and the further removal of acetate salt from the bulk by gel-exclusion chromatography on Sephadex G-50 preequilibrated with 170 mM sodium sulfate. A proper amount of AE dissolved in DMSO was then added to the liposome suspension to obtain a 1:20 AE/lipid molar ratio. AE was solubilized in DMSO because of its scarce solubility in water. The organic solvent added was ≤1% of the total volume, thus having a negligible effect on the liposomes. Liposomes were incubated in the presence of AE for 1 h under conditions of high permeability of the liposome membrane: at 60 °C in the case of DPPC/Chol, DPPC/1/ Chol and at 30 °C in the case of DOPC/1/Chol, POPC/1/Chol formulations. The removal of unentrapped AE was performed by filtration on Sephadex G-50 minicolumns, preequilibrated with 170 mM sodium sulfate, using the dry filtration protocol.28 Evaluation of the Encapsulation Efficacy, E.E. Equal volumes (200 μL) of liposome suspension before and after removal of free AE were dried and then dissolved in 3 mL of DMSO. The E.E. was evaluated by comparing the fluorescence emission of samples at 595 nm (λexc = 432 nm) before (Ib) and after (Ia) the removal of AE, i.e. (Ia/Ib) × 100.
(2)
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where ε and η are the dielectric constant and the viscosity of the medium, respectively, ζ is the zeta potential, and κa is Henry’s function that in the case of an aqueous solution and moderate electrolyte concentration is approximated to 1.5. The values of the zeta potential reported in Table 2 correspond to the average values over several measurements on three independent liposome samples. Surface Potential Measurements. 4-Heptadecyl-7-hydroxycoumarin (C17-HC)-containing liposomes were prepared by adding the proper amount of C17-HC in CHCl3 to the lipid chloroform solution to obtain, after hydration, 50 μM C17-HC. Surface potential measurements were performed on DMPC, DPPC, DOPC, and POPC liposomes, a mixed formulation containing a phospholipid (DMPC, DPPC, DOPC, or POPC), the gemini component and cholesterol in a 1.6:0.4:1 molar ratio. In all samples, the lipid/C17-HC molar ratio was 250:1, and the total lipid concentration was 0.5 mM. The preparation of C17-HC-containing liposomes was carried out using the dry lipid film procedure described above, in the dark to avoid C17-HC photodegradation. Dry lipid films were hydrated using a 170 mM calcium acetate solution at pH 6. Fluorescence excitation spectra of C17-HC were recorded at 25 °C by scanning at the excitation wavelength between 300 and 400 nm at an emission wavelength of 450 nm. The extent of dissociation of C17HC was monitored by the ratio of the excitation fluorescence intensities at 380 and 330 nm. As previously reported,29 the pKa of C17-HC at the water/lipid interface can be described by the following equation pK a = pKH + ΔpK pol + ΔpKel
RESULTS AND DISCUSSION Characterization of AE. To the best of our knowledge, despite the large number of studies on the therapeutic activity of AE, a full physicochemical characterization of AE is absent in the literature. Therefore, we carried out a study on the solubility and the acid−base and spectroscopic properties of AE in different media because this knowledge was necessary both to optimize the loading procedure of AE and to choose the appropriate method for the evaluation of the entrapment efficiency of the compound into liposomes. The solubility and the spectroscopic properties of AE were investigated in organic solvents (isopropanol, DMSO, and chloroform) and in an aqueous medium at different pH values. (UV−vis and emission spectra of AE in DMSO are reported as SI.) AE is scarcely soluble in PBS at pH 7.4, i.e., 19 μM, and gave a colorless solution. The solubility of AE was found to be higher under alkaline conditions; in fact, 1.9 mM AE was completely soluble in 1 M NaOH (pH 14), and the solution assumed a red color. (UV−vis spectra of AE in PBS at pH 7.4 and 14 are reported as SI.) The plot of the absorbance at 504 nm versus pH (spectra and titration curve are reported as SI) allowed us to measure a pKa of 9.7. Note that by UV−vis titration it was not possible to detect the pKa values of the two dissociations. With the aim of loading AE into the internal aqueous compartment of liposomes to exploit the acetate gradient method,17 we also investigated the solubility of AE in the presence of calcium acetate under different pH conditions and found that at pH >8 AE is scarcely soluble in the presence of calcium acetate. In addition, the colorless solutions of AE at pH 8 turn red in the presence of calcium acetate, thus suggesting a dissociated state of AE under these conditions. (UV−vis spectra of AE at pH 8.2 recorded at increasing concentrations of calcium ion and a plot of the absorbance at 504 nm versus the calcium ion concentration are reported as SI.) Therefore, according to the acetate gradient method,13 an efficient active loading of AE into liposomes can occur in the presence of calcium ions at an internal pH of ∼8. Characterization of Liposome Formulations. All formulations investigated for the remote loading of AE into liposomes were characterized with respect to their vesicle size distribution, zeta and surface potentials, and internal pH. The size and size distribution of the lipid aggregates were measured by DLS experiments before the generation of the
(3)
where pKH is the intrinsic proton binding constant, ΔpKpol is the shift in pKa due to a change in surface polarity (dielectric constant), and ΔpKel is the shift in pKa due to a change in the surface potential. The ΔpKel is related to the electrical surface potential by a conversion and rearrangement of the Boltzmann equation29 and can be expressed as ΔpKelk T ln 10 (pK acharged − pK aneutral)kBT ln 10 =− e e (4) where kB is the Boltzmann constant, T is the absolute temperature, e is the electron charge, and pKacharged and pKaneutral are the pKa values in charged lipid bilayers and in neutral lipid bilayers, respectively. The values of the surface potential reported in Table 2 correspond to the average of two independent liposome samples. Inclusion of AE in the Liposome Internal Aqueous Compartment by Remote Loading. Total lipid formulations (12 mM) containing natural phospholipid DPPC, DOPC, or POPC, gemini cationic surfactant 1, and cholesterol (2:1 molar ratio in the case of DOPC/Chol and 1.6:0.4:1 in the case of the phospholipid/1/Chol formulations) featuring a stable transmembrane gradient were φ0HC = −
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DOI: 10.1021/la5038074 Langmuir 2015, 31, 76−82
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Langmuir concentration gradient. The results are reported in Table 1, where the size of the vesicles containing the gemini surfactant
Table 2. Zeta Potential and Surface Potential of Liposomes lipid composition
Table 1. Hydrodynamic Diameter, dH, of Vesicles (170 mM Calcium Acetate, pH 6) lipid composition
dH (nm)a
DPPC DPPC/Chol (2:1) DPPC/1/Chol (1.6:0.4:1) DMPC DMPC/Chol (2:1) DMPC/1/Chol (1.6:0.4:1) DOPC DOPC/Chol (2:1) DOPC/1/Chol (1.6:0.4:1) POPC POPC/Chol (2:1) POPC/1/Chol (1.6:0.4:1)
156 164 159 110 119 457 (52%), 136 (48%) 112 119 106 112 107 106
DPPC DPPC/1/Chol (1.6:0.4:1) DMPC DMPC/1/Chol (1.6:0.4:1) POPC POPC/1/Chol (1.6:0.4:1) DOPC DOPC/1/Chol (1.6:0.4:1)
zeta potential (mV)b
C17-HC pKaa
surface potential (mV)b
20 60
8.7 7.40
77
19 51
8.87 7.92
49
20 45
9.25 8.45
47
22 52
9.10 8.47
37
a
The pKa values of C17-HC were obtained by titration of the probe in the lipid bilayer of different liposome formulations and the corresponding values of surface potential obtained by eq 3. bReported values were averaged values over several measurements, and the error is 5%.
a
The hydrodynamic diameter, dH, of the vesicles was determined by DLS. DH values correspond to the positions of the peaks in the diameter distributions averaged over several measurements (error in determination is 5%) and were obtained from intensity-weighted distributions. The values corresponding to different peaks, together with the intensity-weighted percentages in parentheses, are reported in the case of multimodal distribution.
values are suggestive of a different localization of the probe in the explored formulations. In fact, the values of pKa of C17-HC embedded in neutral liposomes composed of the mere phospholipids are different; in particular, the pKa of the probe in vesicles of saturated phospholipids (DPPC, DMPC) are lower with respect to the pKa measured in vesicles of unsaturated lipids (DOPC, POPC). The minor apparent acidity of the probe in unsaturated vesicles suggests that its position in POPC and DOPC vesicles is not as close to the surface and to cationic headgroups with respect to the position in DPPC and DMPC. Moreover, C17-HC displays a higher pKa in POPC vesicles than in fully unsaturated DOPC vesicles, suggesting a deeper localization in the POPC bilayer. The minor acidity of the probe in vesicles containing unsaturated lipids is also observed in the cationic formulations, though the differences observed between POPC and DOPC vesicles are canceled out in the cationic formulations because the pKa of the probe is very similar in POPC/1/Chol and DOPC/1/Chol vesicles. (Obviously, the surface potential values are different because they are obtained with respect to the neutral POPC and DOPC vesicles.) Internal pH of Liposomes and Creation of the Transmembrane pH Gradient. To load AE ito the internal aqueous compartment of liposomes by the remote loading method, it is first necessary to create a pH imbalance between the internal compartment of liposomes and the bulk and to verify that the gradient is stable. Thus, we explored which of the mixed formulations (1.6:0.4:1 phospholipid/gemini/Chol) were able to maintain a pH gradient across the lipid bilayer. The liposome suspensions were prepared by the hydration of a dry lipid film with 170 mM calcium acetate solution at pH 6 (Experimental Section). According to the acetate gradient method,17 the pH gradient was created by removing the external calcium acetate by gel filtration. The migration of acetic acid (neutral) from the internal aqueous compartment to the bulk across the lipid bilayer increases the internal pH. The internal pH was then measured experimentally using a membrane-impermeant, pH-sensitive fluorescent molecule, pyranine as described in the Experimental Section.17 Note that the pH value measured in the internal aqueous compartment of liposomes before gel filtration, i.e., under equal calcium acetate concentration conditions in the aqueous core of lipid vesicles and in the bulk (Cin = Cout) was, within
are compared to those containing the mere phospholipid, or phospholipid and cholesterol (at 2:1 molar ratio), to evaluate the effect of the presence of gemini on the lipid organization. Liposomes containing unsaturated phospholipids DOPC and POPC were monodisperse with a ∼100 nm hydrodynamic diameter, in agreement with the size imposed by the extrusion procedure. The inclusion in the lipid bilayer of the gemini component or cholesterol, or both of them, did not affect the size of the aggregates. In the case of liposomes containing saturated phospholipid DPPC, the aggregates were larger than expected, featuring a hydrodynamic diameter of ∼160 nm. In the case of DMPC and DMPC/Chol formulations, vesicles can be considered to be monodisperse and in agreement with the size imposed by extrusion. However, upon inclusion of the gemini, DMPC-based vesicles showed a bimodal size distribution of 457 and 136 nm. The results of DLS measurements indicate that the fluidity of the lipid bilayer, determined by the saturation/unsaturation of alkyl chains, controls its capability to interact with the gemini component and with cholesterol and ultimately the morphology of the lipid vesicles. Zeta potential values of liposomes, reported in Table 2, were obtained by electrophoretic mobility measurements. Zeta potential values of vesicles composed of the mere phospholipid are, as expected, slightly positive and equal to each other, with the headgroup of the lipid being the same. Compared to these values, those of liposomes containing cholesterol and gemini 1 are more positive and are quite similar for all of the explored formulations. The surface potential of the cationic liposome formulations was indirectly determined by measuring the pKa of probe 4heptadecyl-7-hydroxycoumarin, C17-HC, embedded in the bilayer, as described in Materials and Methods and evaluated with respect to the neutral formulations containing the mere phospholipid. All of the investigated cationic formulations feature, as expected, a positive ψ0; however, the obtained pKa 79
DOI: 10.1021/la5038074 Langmuir 2015, 31, 76−82
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Langmuir Table 3. Values of the Inner pHa of Liposomes and Stability of the pH Gradient over Time lipid composition DPPC/Chol (2:1) DPPC/1/Chol (1.6:0.4:1) DOPC/1/Chol (1.6:0.4:1) POPC/1/Chol (1.6:0.4:1) DMPC/1/Chol (1.6:0.4:1)
pH0 (R0)
pH24 (R24)
pH48 (R48)
pH72 (R72)
stability (h)
8.1 8.3 8.1 8.5 7.3
8.0 8.0 7.9 8.1
7.8 7.8 7.1 6.6
7.5 (1.66) 7.4 (1.50)
48 48 24 24
