Langmuir 2008, 24, 6965-6980
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Physicochemical and Biological Characterization of Ceramide-Containing Liposomes: Paving the Way to Ceramide Therapeutic Application Elena Khazanov, Aba Priev, Joris P. Shillemans,† and Yechezkel Barenholz* Laboratory of Membrane and Liposome Research, Department of Biochemistry, The Hebrew UniVersity-Hadassah Medical School, POB 12272, Jerusalem 91120, Israel ReceiVed January 22, 2008. ReVised Manuscript ReceiVed March 3, 2008 Ceramides mediate antiproliferative responses, and it has been proposed that increasing the level of ceramides in cancer cells may have a therapeutic antitumor effect. However, ceramides, because of their high ″packing parameter″ (PP), do not form lipid assemblies that can be dispersed in a form suitable for intravenous administration. We found that nanoliposomes containing short- or medium-chain ceramides are unstable because of their very high (>1.3) PP. To overcome this major obstacle, we included the lipopolymer 2kPEG-DSPE, which reduces the additive PP. The presence of PEG-DSPE allows the formation of highly stable (>1 year) ceramide (Cer)-containing nanoliposomes suitable for systemic administration. Using tumor cell lines, we found that the ceramide cytotoxicity was not impaired by their inclusion in nanoliposomes. The use of 14C-labeled ceramides shows that the C6Cer, but not C16Cer, was transferred from the nanoliposomes to the cells and metabolized efficiently. The difference between the two ceramides is related to the large difference between their critical aggregation concentration and was correlated with the much higher cytotoxity of liposomal C6Cer. The activity of 2kPEG-DSPE as a steric stabilizer (as previously shown for Doxil) was also confirmed for C6Cer-containing nanoliposomes. The 2kPEG-DSPE lipopolymer significantly reduced the desorption rate of the ceramide from the liposome bilayer, thereby allowing liposomes containing C6Cer to reach the tumor site and to demonstrate therapeutic efficacy.
1. Introduction A growing body of literature on response to cellular stress is focused on the function of sphingolipids, suggesting that ceramide-related pathways are a fertile area for identification of therapeutic targets.1–4 Ceramides are involved in important aspects of cellular responses to both receptor-dependent and independent stimuli.3 Moreover, attenuation of ceramide levels is increasingly implicated in various stages of cancer pathogenesis. Many studies have reported that various types of cancer cells and tumors can be killed by treatments that increase their ceramide (Cer) level.5,6 It was found that ceramide reduces the activity of telomerase, which is elevated in cancer cells.5,6 Also, both in vitro and in vivo administration of a high dose of B13 (a ceramidase inhibitor, which elevates ceramide levels in cells) was toxic to cancer cells but not to normal hepatic cells.7,8 The main obstacle to therapeutic application of ceramides resides in their physicochemical properties. All ceramides have * Corresponding author. Tel: 972-2-6758507. Fax: 972-2-6757499. E-mail:
[email protected]. † Present address: Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, POB 80 082, 3508 TB Utrecht, Netherlands.
(1) Radin, N. S. Eksp. Onkol. 2004, 26, 3–10. (2) Grassme, H.; Riethmuller, J.; Gulbins, E. Prog. Lipid Res. 2007, 46, 161– 170. (3) Vento, R.; Giuliano, M.; Lauricella, M.; Carabillo`, M.; Di Liberto, D.; Tesoriere, G. Mol. Cell. Biochem. 1998, 185, 7–15. (4) Ogretmen, B.; Schady, D.; Usta, J.; Wood, R.; Kraveka, J. M.; Luberto, C.; Birbes, H.; Hannun, Y. A.; Obeid, L. M. J. Biol. Chem. 2001, 276, 24901– 24910. (5) Sundararaj, K. P.; Wood, R. E.; Ponnusamy, S.; Salas, A. M.; Szulc, Z.; Bielawska, A.; Obeid, L. M.; Hannun, Y. A.; Ogretmen, B. J. Biol. Chem. 2004, 279, 6152–6162. (6) Selzner, M.; Bielawska, A.; Morse, M. A. ; Ru¨diger; H, A.; Sindram, D.; Hannun, Y. A.; Clavien, P. A. Cancer Res. 2001, 61, 1233–1240. (7) Samsel, L.; Zaidel, G.; Drumgoole, H. M.; Jelovac, D.; Drachenberg, C.; Rhee, J. G.; Brodie, A. M.; Bielawska, A.; Smyth, M. J. Prostate 2004, 58, 382–393. (8) Radin, N. S. Biochem. J. 2003, 371, 2432–56.
a very small polar (hydrophilic) headgroup. Regarding the apolar (hydrophobic) region, in the naturally occurring long-chain (C16-24) ceramides, the ratio of the cross-sectional area of the hydrophobic region to the hydrophilic headgroup region is high, which makes them indispersible in aqueous phase, and, therefore, these ceramides are not applicable for intravenous (i.v.) administration. Short-chain (C2, C4) and medium-chain (C6, C8) N-acyl ceramides, which are referred to as “soluble”, were introduced to overcome the above problems of naturally occurring ceramides. However, in spite of their shorter N-acyl chain and therefore their smaller hydrophobic region, they are still not dispersible in a stable way in aqueous phase. Therefore, their in vivo application is restricted. Also, as will be demonstrated in this study, the short- and medium-chain ceramides have poor miscibility with phospholipids (PL), as the assemblies formed from mixtures of a liposome-forming phosphatidylcholine (PC) and ceramide at high mole ratio show distinct phase separation and instability with storage time. Therefore, it is a challenge to design nanoliposomes that include a therapeutically significant amount of ceramides and can deliver them to the tumor sites by passive targeting as a result of the unique microanatomy of tumor vasculature.9,10 Liposomes have a number of properties that make them a versatile carrier for a broad spectrum of hydrophobic, amphipathic, and hydrophilic agents, including drugs, peptides, proteins, plasmid DNA, antisense ODN, and siRNA.11–16 Ceramides are (9) Gregoriadis, G. J. Antimicrob. Chemother. 1991, 28, 39–48. (10) Gregoriadis, G.; Florence, A. T. Cancer Cells 1991, 3, 144–146. (11) Barenholz, Y., Lasic, D. D., Eds. Handbook of Nonmedical Applications of Liposomes; CRC Press: Boca Raton, FL, 1996; Vol. 3. (12) Janoff, A. Liposomes: Rational Design; Marcel Dekker: New York, 1999. (13) Lasic D. D.; Papahadjopoulus, D. Medical Applications of Liposomes; Elsevier: Amsterdam, 1998. (14) Langner, M.; Kral, T. E. Pol. J. Pharmacol. 1999, 51, 211–222. (15) Langner, M. Pol. J. Pharmacol. 2000, 52, 3–14.
10.1021/la800207z CCC: $40.75 2008 American Chemical Society Published on Web 05/30/2008
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hydrophobic molecules and therefore may be suitable candidates for loading into liposomal membranes. Formation of stable liposomes may be predicted from the additive packing parameter (APP) of their components, which is defined as the mole-ratio-weighted sum of the individual packing parameters (PPs) of each of their components. The individual PP is defined as the ratio between the cross-sectional areas of their hydrophobic and hydrophilic regions.17 This concept is explained by the thermodynamics that relates to the hydrophobic effect.18 Stable liposomes are formed when the APP is in the range of 0.74-1.0.19 Among PLs used as liposome-forming lipids, the most common are PCs.20,21 Such PCs have two long acyl chains forming the hydrophobic region of the molecule and a phosphocholine hydrophilic headgroup. For liposome-forming lipids, the cross-sectional areas of these two regions are similar, and therefore such molecules, having a cylinder-like geometry, form stable liposomes. Ceramides, due to their very small and poorly hydrated headgroup, have a PP greater than 1.2 and an inverted-cone-shaped geometry and form hexagonal lattices in lipid monolayers.22,23 Therefore this led us to the working hypothesis that this high PP interferes with the formation of PC-based liposomes containing significant levels of ceramides. This working hypothesis was further supported by the recent observation that cholesterol (PP 1.21),19,24 “competes” with ceramides in lipid bilayers.25 Therefore, we predicted that, on the basis of the concept of APP,19,24 adding to the PC and ceramide an additional component having a low PP would reduce the liposome APP and permit achieving stable ceramide-containing liposomes. Another obligatory condition is that the added component should have a low enough desorption rate (koff) from the liposome lipid bilayer so that liposomes reach the desired site still “loaded” with enough ceramide. On the basis of these two criteria, we used the lipopolymer 2000-Da N-carbamyl-poly-(ethylene glycol methyl ether)-1,2distearoyl-sn-glycero-3-phosphoethanolamine (2kPEG-DSPE) as the APP reducer of choice. This lipopolymer also has the wellestablished effect of sterically stabilizing the liposomes, which is highly advantageous for a carrier of drugs for treating tumors and inflammations.26–29 2kPEG-DSPE, when present in brush conformation, has a PP of about 0.5,24 which reflects its large, highly hydrated headgroup. This PP means that 2kPEG-DSPE has a cone-shaped structure, which complements that of ceramide. Space-filling models of bilayers formed from EPC and 2kPEG(16) de Lima, M. C.; Simo˜es, S.; Pires, P.; Gaspar, R.; Slepushkin, V.; Du¨zgu¨ne, N. Mol. Membr. Biol. 1999, 16, 103–109. (17) Israelachvili, J. N.; Marcelja, S.; Horn, R. G. Q. ReV. Biophys. 1980, 13, 121–200. (18) Israelachvili, J. N. Intermolecular and Surface Forces, 2nd ed.; Academic Press: London, 1992; Chapter 17. (19) Kumar, V. V. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 444–448. (20) Barenholz, Y.; Cevc, G. In Physical Chemistry of Biological Surfaces; Baszkin, A., Norde, W., Eds.; Marcel Dekker: New York, 2000; pp 171-241. (21) Mouritsen, O. G.; Jorgensen, K. Chem. Phys. Lipids 1994, 73, 3–25. (22) Veiga, M. P.; Arrondo, J. L.; Go˜ni, F. M.; Alonso, A. Biophys. J. 1999, 76, 342–350. (23) Hartel, S.; Fanani, M. L.; Maggio, B. Biophys. J. 2005, 88, 287–304. (24) Garbuzenko, O.; Barenholz, Y.; Priev, A. Chem. Phys. Lipids 2005, 135, 117–129. (25) London, E.; London, M. J. Biol. Chem. 2004, 11, 9997–10004. (26) Barenholz, Y. Curr. Opin. Colloid Interface Sci. 2001, 6, 66–77. (27) Gabizon, A.; Shmeeda, H.; Barenholz, Y. Clin. Pharmacokinet. 2003, 42, 419–436. (28) Barenholz, Y. In Liposome Technology; Gregoriadis, G., Ed.; Informa Healthcare: New York, 2007; Vol. 2, pp 1-26. (29) Avnir, Y.; Ulmansky, R.; Wasserman, V.; Even-Chen, S.; Broyer, M.; Barenholz, Y.; Naparstek, Y. Arthritis Rheum. 2008, 58, 119–129. (30) Barenholz, Y.; Amsalem, S. In Liposome Technology. Gregoriadis, G., ed.; CRC Press: Boca Raton, FL, 1993; Vol. 1, p 527. (31) Shmeeda, H.; Even-Chen, S.; Honen, R.; Cohen, R.; Weintraub, C.; Barenholz, Y. Methods Enzymol. 2003, 367, 272–292.
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DSPE and containing C2Cer, C6Cer, or C16Cer (CS Chem3D software, Cambridge, MA) are shown below.
In this study, the physicochemical and structural properties of different ceramides were evaluated. Formation and optimization of liposomes containing various ceramides in the absence and presence of 2kPEG-DSPE were investigated. The liposomal formulations were evaluated for lipid composition and its effect on thermotropic behavior, on bilayer capacity to include and retain ceramides upon long-term storage, and on liposome stability. Different liposomal formulations were evaluated for cellular uptake and metabolism, as well as cytotoxicity and mechanism of cell death, in various cancer cell lines. Moreover, their toxicity and therapeutic activity were evaluated in tumorbearing mice.
Characterization of Ceramide-Containing Liposomes
2. Experimental Section 2.1. Materials. Egg phosphatidylcholine (EPC, Tm ) -5 °C) and fully hydrogenated soybean phosphatidylcholine (HSPC, Tm ) 52.5 °C) were obtained from Lipoid KG (Ludwigshafen, Germany). Cholesterol was purchased from Sigma. N-carbamyl-poly-(ethylene glycol methyl ether)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine triethyl ammonium salt, referred to as 2kPEG-DSPE, (the polyethylene glycol moiety having a molecular weight of 2000 Da), was obtained from Genzyme (Liestal, Switzerland). N-Acetyl-D-erythro-sphingosine (C2Cer), N-tetranoyl-D-erythrosphingosine (C4Cer), N-hexanoyl-D-erythro-sphingosine (C6Cer), N-octanoyl-D-erythro-sphingosine (C8Cer), and N-palmitoyl-Derythro-sphingosine (C16Cer) were obtained from Bio-Lab, Ltd. (Jerusalem, Israel). Dipalmitoyl PC [choline-methyl 3H], [14C]C6Cer, and [14C]C16Cer were purchased from American Radiolabeled Chemicals (St. Louis, MO). All lipids when analyzed by thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC) showed a purity of g97%. tert-Butanol was purchased from BDH (Poole, U.K.) and ethanol (AR) was purchased from Sigma. Water was purified using the WaterPro PS HPLC/Ultrafilter Hybrid model (Labconco, Kansas City, MO), providing sterile, pyrogen-free, highly pure water with low levels of total organic carbon and inorganic ions (18.2 MΩ), which is referred to here as “pure water”. 2.2. Partition between the Two Phases of the Dole System. The Dole two-phase extraction system extracts lipids from plasma and tissue and, at the same time, separates polar lipids (such as PCs and sphingolipids) from apolar lipids (such as cholesterol, cholesterol esters, long acyl chain ceramides, and tri- and acylglycerols).30 Distribution between the two phases (apolar and polar) of the Dole system of ceramides, liposome-forming PCs, and the 2kPEGDSPE lipopolymer were determined as described previously30 and used to calculate partition coefficients. The distribution of each of the lipids between the two phases was measured by quantitative TLC (see Liposome Characterization, below). The concentration of the total PLs, which include 2kPEG-DSPE and either EPC or HSPC, was verified by lipid organic phosphorus content determination using the modified Bartlett method.31 The partition coefficient (Kp) is defined as the ratio of the equilibrium concentration [C] of analyte in the less polar heptanerich Dole upper phase (Dole up) to that in the more polar water- and isopropanol-rich Dole lower phase (Dolelp):
Kp ) [C]Doleup/[C]Dolelp 2.3. Lipid Monolayer Studies and Determination of the Critical Aggregation Concentration of the Ceramides. Various ceramides were dried in vacuum overnight, weighed, and dissolved in hexane/isopropanol (3:2 v/v) to make stock solutions. The surface pressure versus molecular area isotherms were obtained at 25 °C on a water subphase (similar isotherms were obtained on 140 mM NaCl). Barrier speed during compression was 20 mm/min for all monolayers. The measurements were done using the µThrougs Kibron System (Helsinki, Finland). Measurements were carried out by Prof. Peter J. Slotte from Abo Academi University, Turku, Finland. Critical aggregation concentration (CAC) of the ceramides (dissolved in ethanol) at room temperature was determined in pure water containing up to 0.3% ethanol by measuring surface tension as a function of ceramide concentration. The surface tension measurements were done using the µThrougs Kibron System. The measurements at increasing concentrations were made until a constant value of surface tension was reached, indicating the CAC.32 2.4. Preparation of Ceramide-Containing Liposomes. The approach described by Haran et al.33 was used. In brief, tert-butanol stock solutions of each of the lipids to be included in the liposomes were prepared. Appropriate amounts of these solutions were mixed and lyophilized. Citrate buffered saline (CBS) (5 mM sodium citrate, (32) Zuidam, N.; Barenholz, Y. Biochim. Biophys. Acta 1997, 1329, 211–222. (33) Haran, G.; Cohen, R.; Bar, L. K.; Barenholz, Y. Biochim. Biophys. Acta 1993, 1151, 201–215.
Langmuir, Vol. 24, No. 13, 2008 6967 130 mM NaCl, pH 7, 285 mOsmol) was added to hydrate the lyophilized lipids to obtain multilamellar liposomes (MLVs). Large unilamellar nanoliposomes (LUV ∼ 100 nm) or small unilamellar nanoliposomes (SUV < 100 nm) were prepared by extrusion of MLVs 10 times through a 0.2-µm-pore-size polycarbonate filter, followed by extrusion 10 times through a 0.1-µm-pore-size filter (Poretics, Livermore, CA) using the extrusion syringe system of Avanti Polar Lipids (Alabaster, AL). All formulations were stored at 4 °C. 2.5. Preparation of Radioactive Liposomes. For the preparation of radioactive liposomes, appropriate amounts of stock solutions of the desired lipids and ceramides in ethanol were made. 14C-Cer (C6 or C16) and 3H-DPPC were added to reach a final specific radioactivity of 1.2 µCi/µmol for the Cer and 0.6 µCi/µmol for the PCs, respectively. The lipids were hydrated to form MLVs by injecting ethanolic lipid solution into CBS to a final ethanol concentration of 10%, followed by continuous bath sonication for 3 min. LUVs were prepared as described above. Ethanol was then removed by dialysis at 4 °C against 200 volumes of CBS (three exchanges) for 30 min each, and the fourth time was performed overnight against 400 volumes of CBS.34 2.6. Liposome Characterization. 2.6.1. Chemical and Physicochemical Characterization. Liposomes were characterized for their particle size distribution by dynamic light-scattering (DLS) using an ALV-NIBS/HPPS particle sizer ALV-Laser from Vertriebsgesellschaft GmbH (Langen, Germany). Ceramides were quantified by TLC using silica gel plates 60 F254 (Merck, Darmstadt, Germany) in a solvent system of chloroform/ methanol (95:5, v/v). Ceramides and 2kPEG-DSPE were resolved from PCs on the plate using a solvent system of chloroform/methanol/ water (90:15:2.5, v/v/v). After separation, plates were dried and sprayed with copper sulfate reagent (85%), dissolved in 600 mL of pure water. After heating, lipids appeared as dark brown spots. The lipid spot absorbance was quantified using a Fluor-S-MultiImager (Bio-Rad, Hercules, CA). The absorbance level was proportional to the amount of lipid applied to the TLC. The quantity of lipids in each phase was calculated from a standard curve of the appropriate lipid processed by the same procedure described above using TLC. The concentration of total PLs, which include PC and 2kPEGDSPE, was verified by determination of lipid phosphorus by the modified Bartlett method.31 2.6.2. Determination of Maximal LeVel of Ceramide “Encapsulation” into the Liposomes. MLV dispersions with different mole % of C6Cer were centrifuged at 10 000 rpm for 10 min. The pellet, which contains MLVs and other aggregates, and the supernatant, which includes liposomes and micelles, were collected and analyzed for ceramide/PL mole ratio. MLVs were downsized by repeated extrusion as described above, followed by centrifugation at 10 000 rpm for 10 min to remove large particles. The supernatant, referred to as “LUV”, (including LUV, SUV, and micelles) was collected and analyzed for ceramide/PL mole ratio and particle size distribution. TLC was used for quantification of ceramides, and the modified Bartlett organic phosphorus method was used for determination of PL concentration. 2.6.3. Characterization of the Thermotropic BehaVior of the Liposomes. The thermotropic behavior of HSPC-based liposomes (since the Tm of HSPC is 52.5 °C) having different mole % of ceramides and 2kPEG-DSPE were studied using two approaches: differential scanning turbidity (DST) determined as differential temperature scanning of the optical density24,35 and differential scanning calorimetry (DSC).36 DSC measurements were performed on MLVs using a Mettler Thermal Analyzer model 4000. Parameters obtained from DSC measurements include the temperature of maximum change in heat capacity and the enthalpy change of the phase transition. Temperaturedependent changes in specific turbidity (OD/mg lipid) were (34) Peleg-Shulman, T.; Gibson, D.; Cohen, R.; Abra, R.; Barenholz, Y. Biochim. Biophys. Acta 2001, 1510, 278–291. (35) Belsito, S.; Bartucci, R.; Sportelli, L. Biophys. Chem. 2001, 93, 11–22. (36) Biltonen, R.; Lichtenberg, D. Chem. Phys. Lipids 1993, 64, 129–142.
6968 Langmuir, Vol. 24, No. 13, 2008 determined using a Cary 300 Bio UV-visible double beam spectrophotometer (Varian, Australia). The change in OD during temperature scanning relates to the differences in bilayer packing and can be used to monitor phase transition of the bilayer, as previously demonstrated24 and confirmed by the studies presented here. The DST measurements gave a thermogram similar to that obtained by DSC. DST scans were analyzed in two ways: (a) OD as a function of temperature, and (b) d(OD)/dT as a function of temperature. 2.6.4. Density,UltrasonicVelocity,AndCompressibilityMeasurements. The density and ultrasonic velocity of the liposome suspension were determined as previously described24,37 and were used to calculate partial specific volume and adiabatic compressibility.38 2.6.5. Determination of Liposome Stability upon Storage (in Buffer) and in Biological Fluids. Chemical stability of the liposome components was examined by measurement of PL acylester hydrolysis. For this we followed the increase in the level of nonesterified (free) fatty acids (NEFAs) released during storage time.31 The pH of the liposomes during their storage was also followed. Physical stability of the liposomes during storage was followed by time-dependent changes in (i) liposome size distribution as determined using DLS,30 and (ii) level of “free” ceramide by separation of the liposomes (LUVs) from the precipitate (nondispersable lipids), and analysis of lipid composition of both LUVs and precipitate by TLC, as described above. For determination of the physical stability of the liposomes containing ceramides in the presence of serum, we used (i) measurement of changes in liposome particle size distribution by DLS at 25 °C, as described above, and (ii) measurement of turbidity ratio (TR) of LUVs determined spectrophotometrically. For particles having size 1/20 of the wavelength (λ) or smaller (Rayleigh scatterers), it was expected that
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where TR is the turbidity ratio (described as the ratio of absorbances at two different wavelengths). We used the absorbance ratio at λ1 ) 300 and λ2 ) 600 nm. Change of TR with wavelength is an indication of particle size heterogeneity.31 Briefly, LUVs of various defined compositions were incubated for 10 min with 25% or 50% fetal calf serum (FCS) (Biological Industries Beit-HaEmek, Israel) and then measured spectrophotometrically. 2.7. Biological Characterization. 2.7.1. In Vitro Cytotoxicity Studies. Two tumor cell lines in monolayers were used: a human ovarian carcinoma cell line (OV-1063) and a murine colon carcinoma cell line (C-26). Both cell lines were maintained in RPMI-1640 medium supplemented with 10% FCS, antibiotics, and glutamine. All culture medium components were purchased from Biological Industries (Beit-HaEmek, Israel). Cell lines were maintained at 37 °C in a water-jacketed CO2 incubator. The cytotoxicity of liposomal formulations was tested by the methylene blue (MB) staining assay as previously described.39 The net optical density of the MB dye in each well was determined by a plate spectrophotometer (Multiskan Bichromatic Labsystems, Finland) at 620 nm. 2.7.2. Determination of 14C Ceramides and 3H-DPPC Uptake and Metabolism in Cells in Culture. C-26 cells were seeded into six-well plates at a density of 2.5 × 105 in complete medium. The cells were allowed to grow for 48 h and replaced with the supplemented medium. Liposomal or free radiolabeled ceramides were added to the C-26 cells to obtain a final ceramide concentration of 20 µM (7.7 × 104 dpm/32.2 nmol 14C-C6Cer or C16Cer and 2.8 × 105 dpm/217 nmol 3H-DPPC) and incubated for 2, 24, and 48 h at 37 °C. Then the cells were trypsinized and washed with PBS. Cell
lipids, lipids of the medium, and lipids of the wash fraction were extracted by the Bligh and Dyer procedure.40 Two well-separated phases were formed. The water/methanol-rich upper phase was separated from the chloroform-rich lower phase, which contained >99% of the lipids. The lipid-containing lower phase was dried under a nitrogen stream and redissolved in chloroform/methanol (2:1, v/v) ready for analysis by TLC. Analysis was performed using silica gel TLC plates, which were developed in chloroform/methanol/pure water (84:16:1.5, v/v/v). Spots were detected by copper sulfate reagent. The TLC plates were photographed by a Fluor-S-MultiImager (Bio-Rad). Migration of lipids from cell extracts, medium, and wash fractions was visualized in comparison to well-established commercial markers. The retention factors (RFs) of various molecules are as follows: SPM 0.04, EPC or HSPC 0.1, GlcCer 0.4, C6Cer 0.68, and C16Cer 0.88. The TLC plates were then subjected to an imaging plate overnight, and the radioactivity was measured by a Bio-Imaging analyzer (FUJI BAS 1000, Japan). Then the radioactive bands were scraped from the TLC plate and placed into a vial containing Opti-Fluor scintillation medium (Packard Bioscience, Groningen, Netherlands), and the radioactivity was counted by a β-counter (KONTRON Liquid Scintillation Counter). 2.7.3. Assessment of Apoptosis. Tumor cell apoptosis was evaluated by several methods. The morphology of chromatin was assessed by staining with Hoechst-33342, obtained from Calbiochem (La Jolla, CA). This fluorophore preferentially stains dsDNA.41 Briefly, samples containing 5 × 105 cells were cultured on 6-well plates and covered with a glass coverslip. After treatment of cells with IC50 of drugs for 16 h, the cells were washed with PBS and fixed with 4% formaldehyde. Cells were then stained with Hoechst33342 (5 µg/mL) and washed. Thereafter, the glass coverslip was placed on a glass slide, and slides were viewed using a confocal laser scanning microscope (CLSM) (Zeiss 410, Germany), a highresolution microscope that allows viewing and quantification of fluorescence intensity in the different cell compartments. The DNA fragmentation typical of apoptosis was measured by the terminal deoxynucleotide transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) assay (Apoptosis Detection System, Fluorescein, Promega, Madison, WI).42 This method takes advantage of DNA fragmentation during apoptosis and generation of many free 3′-OH termini, which are labeled by fluorescent nucleotides that are enzymatically added to the DNA by TdT. Briefly, OV-1063 cells (3 × 104 cells/mL) and C-26 cells (1.2 × 104 cells/mL) were cultured in a Lab-Tek chambered coverglass system (Nagle Nunc, Naperville, IL) for 48 h. Cells were then treated with IC50 concentrations of the drugs for 24 h, and cellular apoptosis was determined by a CLSM using the TUNEL assay. 2.7.4. EValuation of Toxicity and Antitumor Efficacy of Liposomes Containing Ceramides. All the experimental procedures that make use of animals (mice and dogs) were done in accordance with the standards required by the Institutional Animal Care and Use Committee of the Hebrew University-Hadassah Medical School. The toxicity of liposomes containing ceramides of different N-acyl chain lengths (including C2, C4, C6, C8, and C16) was tested in 8-weekold female BALB/c mice and compared to that of the same liposomes lacking ceramide. To test toxicity, these liposomal formulations at ceramide and lipid concentrations of 2 µmol/mouse and 6 µmol/ mouse, respectively, were injected i.v. three times, at 3-day intervals, and mice weight changes and survival were followed. To test therapeutic efficacy, 8-week-old female BALB/c mice were injected intraperitoneally with 1 × 106 C-26 colon carcinoma cells. The therapeutic efficacies of nanoliposomes containing C6Cer at lipid concentrations of 1-2 µmol/mouse and at 6 µmol/mouse were studied. Treatment began 3 days after tumor cell inoculation
(37) Priev, A.; Zalipsky, S.; Cohen, R.; Barenholz, Y. Langmuir 2002, 18, 612–617. (38) Priev, A.; Almagor, A. ; Yedgar, S. ; Gavish, B. Biochemistry 1996, 35, 2061–2066. (39) Gorodetsky, R.; Levy-Agababa, F.; Mou, X.; Vexler, A. M. Int. J. Cancer 1998, 75, 635–642.
(40) Bligh, E. G.; Dyer, W. J. Can. J. Biochem. Physiol. 1959, 37, 911–917. (41) Jouvet, P.; Rustin, P.; Taylor, D. L.; Pocock, J. M.; Felderhoff-Mueser, U.; Mazarakis, N. D.; Sarraf, C.; Joashi, U.; Kozma, M.; Greenwood, K.; Edwards, A. D.; Mehmet, H. Mol. Biol. Cell 2000, 11, 1919–1932. (42) Gavrieli, Y.; Sherman, Y.; Ben-Sasson, S. A. J. Cell. Biol. 1992, 119, 493–501.
TR ) OD1/OD2 ) (λ2/λ1)4
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Table 1. Distribution and Partition Values of Various Lipids between the Apolar and Polar Dole Phases a % of lipid in % of lipid lipids used in heptane-rich isopropanol/waterrich lower phase in the study upper phase d
C2Cer C4Cer C6Cer C8Cer C16Cer EPC HSPC 2k PEG-DSPE
21.2 ( 4.5 31.5 ( 4.3 62.7 ( 6.1 72.0 ( 5.0 89.0 ( 5.0 15.2 ( 1.4 36.0 ( 3.0 0
78.8 ( 7.1 68.5 ( 5.2 37.3 ( 4.3 28.0 ( 2.0 11.0 ( 2.0 84.8 ( 5.1 64.0 ( 5.0 100
Kpb
liposome formationc
0.54 0.92 3.22 5.12 16.42 0.36 1.13 0
No No No No No Yes Yes No
a The distribution of ceramides was measured by thin-layer chromatography. Kp was calculated as Kp ) [C]Doleup/[C]Dolelp. The concentrations of the 2kPEG-DSPE, EPC, or HSPC were verified by lipid phosphorus content determination.31 For more details, see Experimental Section. b Kp denotes the partition coefficient. c Ability to form liposomes by itself (based on the packing parameter theory). d Cer denotes ceramide.
and was repeated twice for a total of three injections 3 days apart. Each group included eight mice. The median survival and percentage increase in life span of treated (T) over control (C) animals (T/C × 100) were calculated. 2.8. Statistical Analysis. Median survival times and the statistical significance of differences in survival curves were calculated by means of the log-rank test using Prisma Software (GraphPad, San Diego, CA). Differences were considered significant at P < 0.05.
3. Results 3.1. Comparing Hydrophobicity of Ceramides, PCs, and Lipopolymer Used in This Study. Various ceramides (C2Cer, C4Cer, C6Cer, C8Cer, C16Cer), the liposomeforming lipids (HSPC, EPC), and the lipopolymer (2kPEG-DSPE) were first characterized for their distribution between polar (lower) and apolar (upper) phases of the Dole two-phase extraction system. The results are presented in Table 1. The distribution of ceramides was highly dependent on their acyl chain length: the shorter the acyl chain, the smaller the distribution into the apolar heptanerich upper phase (21%, 31%, 63%, 72%, and 89% for C2, C4, C6, C8 and C16Cer, respectively). For the liposome-forming PCs, EPC and HSPC, 15% and 36%, respectively, distributed into the apolar heptane-rich phase. All (100%) of 2kPEG-DSPE was distributed into the polar phase. This is consistent with the high hydration properties of the headgroup of 2kPEG-DSPE.43 The Kp results demonstrate that among all lipids used in this study, the lipopolymer 2kPEG-DSPE is the most polar, in agreement with being the only lipid in the above list that self-assembled as micelles. Interestingly, C2Cer and C4Cer have lower Kp’s than that of the HSPC, i.e., they are more polar than HSPC, while the ceramides having longer acyl chains than that of C4Cer have higher Kp’s than both HSPC and EPC. 3.2. Determination of Critical Aggregation Concentration (CAC) of Ceramides. The ceramides, like almost all amphiphiles, tend to self-aggregate in aqueous phase. The concentration at which this occurs (CAC) was determined by measuring the surface tension as a function of ceramide concentration. C2Cer and C6Cer started to show self-aggregation at concentrations of 8.3 × 10-5 M and 4.4 × 10-5 M, respectively, while C16Cer showed selfaggregation at a much lower concentration of 10-10 M (Table 2). 3.3. Behavior of Ceramides at the Air/Water Interface. We studied the behavior of ceramides that differ in their N-acyl chain lengths at the air/water interface as one of the means to explain their behavior in the liposome membrane. The force/ 2KPEG-DSPE
(43) Tirosh, O.; Barenholz, Y.; Katzhendler, J.; Priev, A. Biophys. J. 1998, 74, 1371–1379.
Table 2. Area Per Molecule of Various Ceramides and CAC of Ceramides and PEG-DSPE a type of lipid
area/molecule (A2) at 20 mN/m2
CAC (M)
C2Cer C4 Cer C6 Cer C8 Cer C16 Cer 2k PEG-DSPE
38 ( 2b 46 ( 2 50 ( 2 45.5 ( 2 37.5 ( 2 n.d.
8.3 × 10-5 n.d.c 4.4 × 10-5 n.d. >10-10 1.2 × 10-5
a The area per molecule of various ceramides at 20 mN/M2 was calculated from the surface-pressure vs molecular area isotherms. The CAC of ceramides and 2kPEG-DSPE was determined by measuring surface tension as a function of lipid concentration. Both measurements were done by using the µThrougs Kibron System. b Unstable monolayer. c n.d., not determined.
area isotherms and area per molecule of these ceramides at a constant pressure of 20 mN/m2 were determined. The monolayer studies show that the C2Cer was the only ceramide from the above list that did not form a stable monolayer at the air/water interface. Areas per molecule of C2 and C4Cer were 38 and 46 A2, respectively. C6Cer has the largest area per molecule of all ceramides tested, 50 ( 2 A2. Starting with C8Cer, the area per molecule began to decrease from 45.5 A2 for C8Cer to 37.5 A2 for C16Cer. 3.4. Size Distribution of Ceramide Dispersions in Various Media. Most ceramides have low to very low aqueous solubility and dispersibility due to their small and poorly hydrated headgroup. The most commonly used means to achieve ceramide dispersion in aqueous phase is to dissolve it in either ethanol or ethanol/dodecane solutions and then dilute it in aqueous medium.44,45 When C2Cer, C6Cer, and C16Cer dissolved in a mixture of ethanol/dodecane (98:2 v/v) were injected into 100 volumes of serum-free medium, a milky, translucent dispersion was formed. Evaluation of the size distribution of these different ceramide dispersions by DLS revealed that the mean diameter of the particles formed by C2Cer and by C6Cer was 330 nm, and that formed by C16Cer was 790 nm. However, when these ceramide dispersions were further diluted 10-fold in 50% serumcontaining medium, a 4-6-fold additional increase in the mean particle size with a much broadened size distribution occurred. Attempts were made to inject ceramide dispersions i.v. One or two micromoles per mouse of C6Cer dispersed in ethanol or in ethanol/dodecane (98:2) were injected i.v. into mice (6 mice per group). The concentration of ethanol in blood was in the range of 3.3-6.7%. Of the six mice injected, two died in each group. No death occurred in the control (vehicle injected) group. That is, death is ceramide, but not ethanol related. The remaining mice were followed at 3-day intervals for two weeks. These mice lost 5-10% of their initial weight compared with the vehiclealone injected group, who gained weight. Therefore we concluded that i.v. injection of ceramide dispersion in ethanol or in ethanol/dodecane was toxic to mice and is not a suitable mode of administration. 3.5. CharacterizationoftheLiposomesContainingCeramides. 3.5.1. Efficiency of Ceramide Inclusion in the Liposomes. The mole ratio of PL (sum of PC and PEG-DSPE) to ceramide in the LUV (output) to this ratio in the lipid mixture used for the liposome preparation (input) was used to calculate efficiency (percent) of inclusion of various ceramides in the liposomes. For example, Table 3 shows that 55-95% of the ceramides used for liposome preparation (compare Nos. 9-25 in column labeled “% of input”) were incorporated into the LUV. We found that the output value depends on the input mole % of ceramide, type of liposome PC, (44) Ji, L.; Zhang, G.; Uematsu, S.; Akahori, Y.; Hirabayashi, Y. FEBS Lett. 1995, 358, 211–214. (45) Luberto, C.; Hannun, Y. Methods Enzymol. 2000, 32, 407–420.
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KhazanoV et al. Table 3. Characterization of LUV Formulations
a
no.
liposome composition
input mole ratio
initial sizeb (nm)
physical state of the membrane at 37°c
actual ceramide % of input
physical stability at 4 °C, month
APP
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
EPC HSPC EPC/2kPEG-DSPE HSPC/2kPEG-DSPE EPC/2kPEG-DSPE HSPC/2kPEG-DSPE EPC/2kPEG-DSPE HSPC/2kPEG-DSPE EPC/2kPEG-DSPE/C2Cer HSPC/2kPEG-DSPE/C2Cer EPC/2kPEG-DSPE/C4Cer HSPC/2kPEG-DSPE/C4Cer EPC/C6Cer EPC/2kPEG-DSPE/C6Cer EPC/2kPEG-DSPE/C6Cer HSPC/C6Cer HSPC/2kPEG-DSPE/C6Cer HSPC/2kPEG-DSPE/C6Cer EPC/2kPEG-DSPE/C6Cer HSPC/2kPEG-DSPE/C6Cer EPC/2kPEG-DSPE/Chol/C6Cer EPC/2kPEG-DSPE/C8Cer HSPC/2kPEG-DSPE/C8Cer EPC/2kPEG-DSPE/C16Cer HSPC/2kPEG-DSPE/C16Cer
100 100 69.5/7.5 69.5/7.5 66/11 66/11 81/7.5 81/7.5 69.5/7.5/23 69.5/7.5/23 69.5/7.5/23 69.5/7.5/23 77/23 69.5/7.5/23 66/11/23 77/23 69.5/7.5/23 69.5/7.5/23 81/7.5/11.5 81/7.5/11.5 44/7.5/37/11.5 69.5/7.5/23 69.5/7.5/23 69.5/7.5/23 69.5/7.5/23
89 92 111 112 85 82 104 84 97 82 91 75 98 98 98 147 99 153 104 84 92 85 86 93 127
LD SO LD SO LD SO LD SO LD SO LD SO LD LD LD SO SO SO LD SO LO LD SO LD SO
94d 75d 88d 85d 100d 100d 96d 90d 65 58 57 55 59 68 95 61 74 74 78 71 75 62 71 65 56
24 24 48 ongoing 48 ongoing 36 36 48 ongoing 48 ongoing 24 4 14 3 3.5 4.5 6 1.25 2 6 24 8 0.25 48 ongoing 48 ongoing 48 ongoing 48 ongoing
0.8 0.82 0.80 0.82 0.77 0.79 0.80 0.82 0.93 0.95 g0.94 g0.96 0.96 0.96 0.98 0.98 0.98 0.97 0.89 0.91 g1.00 g0.91 g0.93 0.91 0.93
a Mole % of ceramide in LUV was determined by TLC. Output of ceramide in LUV was calculated from % of PL recovery (determined by organic phosphorus.31 b Variation in size of liposomes from mean size is about 10% or less. c LD, liquid disordered (fluid phase); SO, solid ordered (gel phase); LO, liquid ordered (fluid phase). d % of PL input.
and the presence and level of 2kPEG-DSPE. For example, in liposomes with a higher (7.7/1) (PL+2kPEG-DSPE)/ceramide ratio (e.g., EPC or HSPC/2kPEG-DSPE/C6Cer (81/7.5/11.5)) the “loading” of C6Cer was higher by about 14% than for lipid assemblies with lower (3.3/1) ratio (EPC or HSPC/2kPEG-DSPE/ C6Cer (69.5/7.5/23)), as shown in Table 3. It was also found that increasing the mole % of the lipopolymer enables one to increase the mole % of ceramide (e.g., C6Cer) in the liposome lipid bilayer (compare Nos. 13-15 in the column labeled “% of input”) and to improve the liposome stability upon storage at 4 °C. 3.5.2. Determination of Maximum Loading Capacity of Ceramides into MLV and LUV Bilayers. We determined the maximum capacity of liposome bilayers composed of either HSPC (“solid”) or EPC (“fluid”) liposome-forming PCs to include ceramides in both MLV and LUV and whether the inclusion was affected by increasing the mole % of the lipopolymer 2kPEGDSPE. It was found that the maximum loading capacity of C6Cer into the liposomes containing 7.5% as well as 11% 2kPEG-DSPE is 35 mol % (data not shown). 3.5.3. Stability of the Liposomes Containing Ceramides upon Storage at 4 °C. When stored at 4 °C in CBS, the liposome formulations described in Table 3 were chemically stable for at least 12 months, as the level of NEFA did not increase above 3%, and the pH remained constant. However, liposomes varied to a large extent in their physical stability, as indicated by following the aggregation and/or fusion of liposomes, changes in their particle size distribution, as well as macroscopic demixing of the liposome components leading to the ceramide separating of the liposome to form a ceramide-rich precipitate. Table 3 summarizes the physical stability of LUVs during storage at 4 °C. It was found that, upon storage at 4 °C, liposomes of certain compositions based on either EPC or HSPC show a satisfactory long-term physical stability of about 4 years, although, in general, EPC-based liposomes show better physical stabilility than HSPCbased liposomes (for example, compare Nos. 13, 14, and 15 to
16, 17, and 18 in Table 3). Furthermore, liposomes containing C2, C4, and, especially, C6Cer in their bilayer were less stable than liposomes having C8 or C16Cer. We found that liposomes containing 2kPEG-DSPE (sterically stabilized liposomes, SSLs) were more physically stable than liposomes lacking 2kPEG-DSPE (compare Nos. 13, 14, and 15 to 16, 17, and 18 in Table 3). When cholesterol was included in the liposomes, for example, a formulation of EPC/Chol/2kPEG-DSPE/C6Cer (44/7.5/37/23 mol ratio), the resulting liposomes were physically unstable and disintegrated within ∼1 week (Table 3). This may be explained by the fact that cholesterol, whose PP is 1.2119 increases the APP, as do ceramides. When comparing the liposomes having the same 23 mol % of the various N-acyl ceramides and 7.5 mol % of 2kPEG-DSPE, the relative stability upon storage at 4 °C was as follows: EPC or HSPC/2kPEG-DSPE/C16Cer (C8Cer) > EPC/2kPEG-DSPE/C2Cer > EPC/2kPEG-DSPE/ C4Cer > EPC/ 2kPEG-DSPE/C Cer > HSPC/2kPEG-DSPE/C Cer > HSPC/ 6 2 2kPEG-DSPE/C Cer > HSPC/2kPEG-DSPE/C Cer. Namely, 4 6 liposomal formulations having medium- or short-chain ceramide have the poorest stability. 3.6. Stability of the Liposomal Formulations in Serum. 3.6.1. Effect of Serum on Liposome Size Distribution. Liposomes are formulated for i.v. administration; therefore, it was important to evaluate the effect of serum on their physical stability. For this, we measured changes in liposome size distribution after their exposure to serum using DLS. It was found that no size changes occurred for nSSLs lacking ceramides or including C2 and C6Cer upon exposure to 50% of FCS. However, nSSLs composed of EPC/PEG-DSPE/C16Cer in FCS increased dramatically in size and size distribution heterogeneity (Table 4). 3.6.2. Turbidity Measurement of Different LUV Formulations in Serum. The advantage of using turbidity measurements is that, unlike DLS, it determines all changes in the system and is not affected by the DLS software, which may exclude certain particle populations.30 In order to evaluate changes in liposome size, we determined the TR, which is the ratio OD300/OD600. The
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Table 4. Influence of Serum on Size Distribution of Various nSSLs a,b size in liposome CBS mean formulations (ceramide mole %) ( SD, nm nSSL nSSL-C2Cer (23) nSSL-C6Cer (23) nSSL-C16Cer (23)
98 ( 5 104 ( 7 108 ( 10 140 ( 5
size in 50% serum mean ( SD, nm 132 ( 15 (unimodal) 112 ( 7 (unimodal) 138 ( 10 (unimodal) 894 (polymodal)
TR in TR in 50% CBS serum 7.96 8.38 7.89 7.59
8.6 7.9 7.7 4.2
a Liposomes were characterized for their particle size distribution by DLS, from which mean size ( SD and type of distribution (uni- or polymodal) was determinated. Turbidity of the dispersions was determined as OD using a spectrophotometer. b SD, standard deviation. TR, turbidity ratio ) T at 300 nm/T at 600 nm. CBS, citrate buffered saline.
results of turbidity measurements confirm those obtained by DLS, and show that only nSSLs containing the long-chain C16Cer increase in size (aggregate) in the presence of serum (Table 4). This aggregation was monitored by the reduction in TR of EPC/ PEG-DSPE/C16Cer nSSLs from 7.59 (without FCS) to 4.2 in the presence of serum. Almost no changes for nSSLs containing either C2 or C6Cer or those lacking ceramides occurred in the presence of FCS. 3.7. Thermotropic Behavior of MLVs and LUVs Containing Various Ceramides. 3.7.1. Thermotropic BehaVior of MLVs Composed of HSPC, 2kPEG-DSPE, and Different Ceramides. Following thermotropic behavior is an effective method for characterizing the miscibility of liposome lipid components in the lipid bilayer. We used two independent methods to characterize thermotropic behavior: DST24,35 and DSC.36 Figure 1A-C describes the curves of DST as dOD/dT versus temperature of MLVs containing increasing amounts of C2, C6, and C16 ceramide, respectively. Figure 1A clearly demonstrates that increasing the mole % of C2Cer leads to an increase in lowering and broadening of the range of the SO-LD phase transition as well as of Tm (the temperature of maximum change in the dOD/dT). The area under the curve is similar up to 50 mol % and is reduced significantly at 75 mol % of C2Cer. The SO to LD phase transition presented in Figure 1B shows that C6Cer has the poorest miscibility with HSPC. This indicated by the findings that C6Cer induces phase separation to HSPCenriched domains (see the lowering of the temperature range of the peak at high temperature) and to C6Cer-enriched domains (represented by the peak at lower temperature). Figure 1C shows that the addition of C16Cer to HSPC shifted upward the Tm and the range of the SO-LD phase transition. This indicates good miscibility between HSPC and C16Cer. Of all the ceramides used, C16Cer was the most miscible with HSPC. 3.7.2. Thermotropic BehaVior of MLVs and LUVs composed of HSPC, 2kPEG-DSPE, and C6Cer. The appearance of a second peak in the DST scan indicates that, in the liposomes containing 25 mol % of C6Cer, phase separation to HSPC-enriched and C6Cer-enriched phases occurs. Therefore, the effect of increasing the mole % of 2kPEG-DSPE on HSPC and C6Cer miscibility was studied by DST measurements. Figure 2A (presenting MLVs) and Figure 2B (presenting LUVs) confirms that two peaks exist in the thermograms of liposomes lacking the lipopolymer. At 5 mol % of lipopolymer, the area under the curve of the second peak (related to the ceramide-enriched phase) was reduced, suggesting reduction in phase separation. Further increasing the mole % of 2kPEG-DSPE to 7.5 and 10 mol % resulted in the disappearance of the second peak in the thermogram, suggesting a lack of phase separation and almost complete miscibility of all the liposome components. The increase of mole % of 2kPEGDSPE to 12.5 and 20 caused a gradual disappearance of the peak at 321 K (HSPC-enriched phase) due to partial bilayer solubi-
lization to form mixed micelles, for which “cooperativity” of the phase transition is low. Micelle formation was also evident from the reduction in mean particle size, as determined by DLS and turbidity measurements (data not shown). The data observed from DST described in Figure 2A,B were in good agreement with results obtained by DSC (Figure 3), in which only one peak (at 321 K) was obtained in the thermogram at 10 mol % of 2kPEG-DSPE. Comparing the thermograms of MLVs to those of LUVs (Figure 2A,B) shows their similarity, indicating that the MLVs are assemblies, which contain all lipid components in the same lipid bilayer. In LUVs composed of HSPC/C6Cer without lipopolymer (Figure 2B), two peaks are distinguished clearly as with the MLVs (Figure 2A), although the relative size of the lowertemperature peak at 308 K is smaller in the MLVs. Apparently, the downsizing (from MLVs to LUVs) of the liposomes caused a decrease in phase separation, producing liposomes (LUVs) in which the immiscibility of the liposome lipid components was lower than that in the MLVs. This is expected for smaller and more curved assemblies.18,35 Namely, downsizing decreases phase separation. These results also demonstrated that the 2kPEG-DSPE improves ceramide miscibility with other bilayer components, irrespective of liposome size and lamellarity. 3.8. Effect of LUV Composition on the Partial Specific Volume and Compressibility of the Liposome Membrane. Packing of various liposomal formulations was determined by partial specific volume and partial specific compressibility studies (calculated from ultrasound velocity and density measurements as described in the Experimental Section). LUVs composed of a mixture of one of the following ceramides, C2Cer, C6Cer, and C16Cer, with one liposome-forming PL (EPC or HSPC), and the lipopolymer 2kPEG-DSPE, were characterized for their partial specific volume and partial specific compressibility. Partial specific volumes, Vj, of liposomes composed of EPC/ Cer (0.77/0.23 mol %) were 0.9888 mL/g for EPC/C2Cer, 1.0005 mL/g for EPC/C6Cer, and 0.9764 mL/g for EPC/C16Cer. The results show that nanoliposomes consisting of C6Cer had the highest partial specific volume. Figure 4A,B shows the influence of PC acyl chain saturation and the presence of 2kPEG-DSPE (7.5 mol %) on the partial specific compressibility of LUVs having C2Cer, C6Cer, and C16Cer. LUVs consisting of unsaturated EPC had a higher compressibility than LUVs composed of the saturated HSPC. This is consistent with the physical state of the membrane at room temperature being predominantly liquid disordered (LD) for EPC-based and solid ordered (SO) for HSPC-based lipid bilayers. Figure 4A,B also presents the changes in compressibility of the liposomes as a result of adding the 7.5 mol % 2kPEGDSPE. The addition of the lipopolymer decreased the compressibility of LUVs composed of either EPC or HSPC, indicating a decrease in defects (related to free volume) in the lipid bilayer, resulting in more tightly packed bilayers for the more saturated liposomes. The results show that LUVs composed of EPC or HSPC (with or without 2kPEG-DSPE) and C6Cer possess a higher compressibility (Figure 4A,B) than those containing C2Cer or C16Cer. The statistical significance for specific compressibility of liposomal formulations as a function of matrix lipid or ceramide (C2, C6, or C16 Cer) used with and without 2kPEG-DSPE was determined by Student’s t test (p < 0.05). Nanoliposomes that include C6Cer are expected to have more free-volume-related defects in the lipid bilayer and be less tightly packed than similar liposomes with other ceramides, as proposed in the structural model. Comparing the effect of various ceramides, liposomes having C16Cer showed the lowest partial specific compressibility
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Figure 1. Temperature-dependent changes of the optical density of HSPC/2kPEG-DSPE (95:5) MLV at different mole % (0, 12.5, 25, 50, or 75) of C2Cer (A), C6Cer (B), or C16Cer (C) as a function of temperature were determined using a Cary 300 Bio UV–visible double-beam spectrophotometer. Temperature-dependent scans were presented as first derivative curves of the optical density (dOD/dT).
(even lower than the EPC-based liposomes lacking ceramides), indicating the smallest free volume and least lipid bilayer defects. 3.9. Cytotoxic Activity of Ceramide-Containing Liposomes against Cancer Cell Lines. Studies of ceramide cytotoxic activity in cell cultures usually employed ethanol dispersions of ceramide. Therefore, in our in vitro study, the cytotoxic activity of ceramides dispersed in ethanol was used as a reference and was compared to that of ceramides in liposomes. Ceramide cytotoxicity was determined by the MB assay (see Experimental Section). We found that all of the tested ceramides that included C2, C4, C6, C8, and C16 when introduced into cell cultures either as ethanol dispersions or as part of liposomes were cytotoxic to a similar extent against C-26 murine colon and OV-1063 human ovarian tumor cell lines; that is, IC50 values were similar (Table 5). Cytotoxic activity was dependent mainly on the type of ceramide used.
In order to better understand the effect of the N-acyl chain length of the ceramides on their cytotoxic activity, cellular uptake and metabolism of liposomal and free ceramides were investigated. 3.10. Cell Uptake and Metabolism Studies. We studied in C-26 cells the relationship between ceramide cytotoxicity and its uptake and metabolism. For this, radiolabeled ceramide N-14Chexanoyl liposomal or non-liposomal (free) C6Cer was used. We followed uptake and metabolism by the cells with time. Uptake of the liposomes themselves was followed using 3H-DPPC (as a marker of liposome-forming lipids). Ceramide biofate was studied by following the metabolism of 14C-C6Cer to form 14C6 sphingomyelin (C6SPM) and 14C-C6 glucocerebroside (GlcCer), which were found in the cells and in the cell growth medium (Figure 5 and Figure 6A,B, respectively). The quantitative aspects of the metabolism of C6Cer in C-26 cells were studied by TLC
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Figure 4. Bar graphs showing the influence of the lipid matrix EPC (A) or HSPC (B) each alone or in combination with different ceramides (C2Cer, C6Cer, or C16Cer) with or without 2kPEG-DSPE, on partial specific compressibility of LUV.
Figure 2. Temperature-dependent changes in the optical density of HSPC/ C6Cer liposomes containing various amounts of 2kPEG-DSPE/MLV (A) or LUV (B) as a function of temperature were determined using a Cary 300 Bio UV–visible double-beam spectrophotometer. Temperaturedependent scans were presented as first derivative curves of the optical density (dOD/dT).
Figure 3. Thermotropic behavior of MLV based on HSPC and C6Cer (3:1) containing various amounts of 2kPEG-DSPE was studied using differential scanning calorimetry (DSC). DSC measurements were performed on MLV using a Mettler thermal analyzer (model 4000). Parameters obtained from DSC measurements include the temperature range of the solid-ordered to liquid-disordered phase transition and the temperature of the maximum change in the heat capacity (Tm).
analysis and by total lipid extracts of cells, and determination of the radioactivity level of 14C ceramide and its metabolites in cells and growth medium. Recovery of total 14C-Cer and 3HDPPC radioactivity was above 75%, giving the data good reliability. Figure 5 is a radiochromatograph of the 14C-C6Cer taken up by the cells and its metabolism resulting in the formation of 14C-C SPM and 14C-C -GlcCer. Lipid extract from cells treated 6 6 for 2, 24, or 48 h with free or nanoliposomal ceramide were analyzed by TLC. Figure 5 clearly shows that both cell uptake and metabolism of 14C-C6Cer increase with time. Liposomal ceramide was taken up more slowly than the free ceramide, and the presence of 2kPEG-DSPE in the liposomes further slowed down both the uptake and metabolism of C6Cer. Table 6 and Figure 6 show a quantitative analysis of the TLC performed as described in the Experimental Section. Figure 6A demonstrates that free and liposomal C6Cer were efficiently taken up and metabolized by C-26 cells in a time-dependent manner. After 2 h of incubation, the uptake of C6Cer was in the following order: free C6Cer > EPC/C6Cer > HSPC/C6Cer > EPC/2kPEGDSPE/C6Cer > HSPC/2kPEG-DSPE/C6Cer. After 24 h incubation, all of the free C6Cer and liposomal EPC/C6Cer were taken up by the C-26 cells (Figure 6B), while 11% of the 14C6Cer was retained in the HSPC-based liposomes (Figure 6B). For both PCs, the presence of 2kPEG-DSPE slows down ceramide uptake by the cells, since 6.5% or 24% of the14C6Cer from EPC/2kPEG-DSPE/C6Cer or from HSPC/2kPEGDSPE/C6Cer liposomes, respectively, remains in the medium. This agrees with the slower release of C6Cer from pegylated nSSLs compared to nonpegylated nanoliposomes (Figure 6B). Analyzing the overall uptake and metabolism of C6Cer presented in Table 6, we found that differences between the uptake of free and liposomal 14C-C6Cer were pronounced mainly at short incubation times (2 h); 39.9%, 28.8%, and 23.6% of C6Cer was taken up and metabolized by cells from the ethanolic dispersion or from non-SSL liposomes based on EPC or HSPC,
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Table 5. Cytotoxic Activity of Liposomal or Non-Liposomal (Free) Ceramides against OV-1063 or C-26 Tumor Cell Lines treatments
IC50 (µM), 4 h
IC50 (µM), 24 h
a
IC50 (µM), 72 h
cells:
OV-1063
C-26
OV-1063
C-26
OV-1063
C-26
C2Cer (in ethanol) EPC/2kPEG-DSPE/C2Cer HSPC/2kPEG-DSPE/C2Cer C4Cer (in ethanol) EPC/2kPEG-DSPE/C4Cer HSPC/2kPEG-DSPE/C4Cer C6Cer (in ethanol) EPC/2kPEG-DSPE/C6Cer HSPC/2kPEG-DSPE/C6Cer C8Cer (in ethanol) EPC/2kPEG-DSPE/C8Cer HSPC/2kPEG-DSPE/C8Cer C16Cer (in ethanol) EPC/2kPEG-DSPE/C16Cer HSPC/2kPEG-DSPE/C16Cer
>80.0 >80.0 >80.0 10.0 ( 2.7 14.0 ( 4.0 25.0 ( 3.0 8.0 ( 1.0 14.0 ( 5.0 15.0 ( 3.0 >40.0 >40.0 >40.0 >100.0 >100.0 >100.0
>60.0 >60.0 >60.0 15.5 ( 3.5 18.0 ( 2.0 17.0 ( 0.9 11.0 ( 5.0 21.0 ( 6.0 18.0 ( 2.8 >40.0 >40.0 >40.0 >100.0 >100.0 >100.0
40.0 ( 6.0 53.0 ( 10.0 70.0 ( 5.0 5.9 ( 0.14 6.5 ( 1.9 13.0 ( 4.0 4.5 ( 0.7 6.7 ( 3.0 7.5 ( 0.7 23.7 ( 1.0 27.0 ( 4.0 31.0 ( 0.8 >100.0 >100.0 >100.0
35.0 ( 5.0 54.0 ( 10.0 80.0 ( 8.0 6.2 ( 0.35 5.5 ( 2.0 8.0 ( 2.0 4.1 ( 1.6 4.75 ( 1.4 7.0 ( 2.8 23.0 ( 1.4 23.0 ( 3.5 43.0 ( 3.1 >100.0 >100.0 >100.0
25.0 ( 1.9 24.0 ( 7.0 25.0 ( 3.0 3.9 ( 0.6 1.5 ( 0.58 3.5 ( 0.5 2.3 ( 1.0 2.5 ( 0.7 4.2 ( 1.7 19.0 ( 1.4 12.5 ( 2.5 12.0 ( 3.9 90.0 ( 5.0 84.0 ( 6.0 98.0 ( 10.0
20.0 ( 1.0 19.0 ( 5.0 32.0 ( 3.0 3.7 ( 0.35 3.2 ( 1.1 4.0 ( 2.0 2.9 ( 1.2 2.0 ( 0.5 3.2 ( 1.7 21.5 ( 2.1 16.2 ( 2.8 16.9 ( 3.4 80.0 ( 6.0 79.0 ( 7.0 100.0 ( 8.0
a
Cytotoxicity was determined by the MB assay.
Figure 5. Uptake of 14C-radio-labeled C6Cer and its metabolites in C-26 cells. C-26 cell extracts were separated on TLC plates, developed in chloroform/methanol/pure water (84/16/1.5 by vol), and then subjected to an imaging plate overnight. The radioactivity was visualized by the Bio-Imaging analyzer after incubation with free or liposomal 14C-C6Cer for 2, 24, or 48 h.
Table 6. C-26 Cell Uptake of 14C-Labeled Liposomal or Non-Liposomal (Free) C6Cer or C16Cer Time:
treatments
2h
24 h
48 h
cell cell cell cell cell initial lipid/Cer molecule uptake cell lipid/Cer uptake uptake lipid/Cer uptake uptake lipid/Cer ratio (mol/mol) followed (nmole) uptake (%) ratio (nmole) (%) ratio (nmole) (%) ratio 14
C6Cer EPC/C6Cer
6.7
EPC/2kPEG-DSPE/C6Cer
6.7
HSPC/C6Cer
6.7
HSPC/2kPEG-DSPE C6Cer
6.7
C16Cer EPC/2kPEG-DSPE/C16Cer
6.7
b
a
C6Cer C6Cer 3 H-DPPC 14 C6Cer 3 H-DPPC 14 C6Cer 3 H-DPPC 14 C6Cer 3 H-DPPC 14 C16Cer 14 C16Cer 3 H-DPPC 14
13.7 7.4 6.4 6.3 5.9 6.0 5.6 4.9 5.2 0.4 0.0 0.0
39.9 28.8 3.3 19.7 2.7 23.6 2.9 19.0 2.7 1.1 0.0 0.0
0.9 0.9 0.9 0.7 0.0
34.5 22.7 20.2 28.2 21.5 17.4 13.8 15.6 9.9 n.d. n.d.
93.7 88.7 10.4 87.6 9.9 67.9 7.1 60.7 5.1 n.d. n.d.
0.8
24.5 20.0 22.5 31.4 3.3 n.d.b
72.0 78.4 11.6 97.4 1.5 n.d.
n.d.
0.6
n.d.
n.d.
n.d.
n.d. n.d.
4.3 0.8 5.0
12.2 2.9 2.9
6.6
0.9 0.8
0.1 0.1
a Cell lipids were extracted by the Bligh and Dyer procedure, separated by TLC, and analyzed by a β-counter as described in the Experimental Section. n.d., not determined.
respectively. In both cases, the presence of 2kPEG-DSPE slows down the uptake and metabolism of C6Cer. Similar uptake and metabolism follow-up studies were performed on formulations containing the long-chain C16Cer. Table 6 also shows that C16Cer was taken up by the cells at a much slower rate than C6Cer. Regarding C16Cer, it also metabolized into C16SPM, but at a much slower rate (8.4% only) than C6Cer, and most of the C16 SPM (in the case of growing cells for 48 h with free C16Cer) remains in cells, as opposed to
C6Cer, where almost all of the C6SPM is released to the cell growth medium (Figure 6B). The differences in uptake of C6Cer and C16Cer also explains the low cytotoxicity of C16Cer against cancer cell lines, as was shown previously (Table 5). In all the above experiments, 3H-DPPC was used as a liposome marker. We found that the uptake of 3H-DPPC was much lower than that of the 14C6Cer. Only 3.3% of 3H-DPPC was taken up by the cells from EPC-based liposomes compared to 28.8% of C6Cer after 2 h (Table 6). The same results were obtained for
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Figure 6. Distribution of radio-labeled C6Cer and its metabolites in cells (A) and in a cell-derived medium (B). Free C6Cer or LUV containing 14C6Cer was incubated with C-26 cells for 2 and 24 h. Total lipids were extracted from cells by the Bligh and Dyer procedure, and the level of radio-labeled C6Cer was determined by the β-counter, as described in Materials and Methods. Free C6Cer–white columns, EPC/C6Cer–dark-grey columns, EPC/ 2k PEG-DSPE/C6Cer–black columns, HSPC/C6Cer–gradient columns, and HSPC/2kPEG-DSPE/C6Cer–light grey columns.
HSPC-based liposomal formulations (Table 6). A similar pattern of lipid uptake occurred after 24 and 48 h (Table 6). This suggests that C6Cer was not taken up by cells as part of liposomes, but rather as free ceramide. 3.11. Assessment of Apoptosis. In order to distinguish between apoptotic and nonapoptotic death (necrosis), the mechanism by which ceramides induce cell death was studied by three different methods. Apoptosis results in chromatin and cytoplasm condensation with a pronounced decrease in cell volume, plasma membrane blebbing, and transformation of the nucleus into membrane-bound apoptotic bodies. All these features were assessed by analysis of cells after staining of cell dsDNA with Hoechst-33342 by a CLSM (Table 7). The results of this staining show that 51% of the OV-1063 cells appeared to be apoptotic after 16 h of treatment with nSSL-C6Cer, compared to only 4% of untreated cells (Table 7). Moreover, the TUNEL method, which measures fragmentation of DNA, showed that a large (∼80%) proportion of OV-1063 cells treated with IC50 concentrations of C6Cer delivered as EPC/ 2kPEG-DSPE/C Cer became apoptotic after 24 h of incubation 6 with the ceramide, in contrast to untreated ones, which showed a lack of DNA fragmentation. Therefore we concluded that there was a positive correlation between all the methods used for the detection of apoptosis in cells treated with ceramides. Table 7 demonstrates a quantitative analysis of the percentage of OV-1063 cells that undergo apoptosis. Our results demonstrated that C2Cer, C4Cer, and C6Cer
Table 7. Induction of Apoptosis in Treated Tumor Cells
a
apoptotic cells (% of total cell number) treatment
16 h
24 h
untreated C2Cer (in ethanol) nSSL-C2Cer C4Cer (in ethanol) nSSL-C4Cer C6Cer (in ethanol) nSSL-C6Cer C16Cer (in ethanol) nSSL-C16Cer
4 48 48 not done not done 53 51 not done not done
3 60 73 58 55 61 59 17 18
a Percent of apoptotic OV-1063 cells after treatment with free or liposomal ceramides as determined by the staining of dsDNA with Hoechst-33342 as described in the Experimental Section.
were more than 3 times as efficacious in the induction of apoptosis as C16Cer, in agreement with the cytotoxicity and cell uptake studies shown in the previous sections. 3.12. Toxicity of Nanoliposomes Containing Ceramides in Tumor-Bearing Mice. In vivo toxicity of the nanoliposomes containing C2, C4, C6, C8, or C16 ceramides based on either EPC or HSPC as liposome-forming lipids was evaluated. Toxicity evaluation, based on following mice appearance, weight, and survival, clearly shows that, at a dose of 1 or 2 µmol of ceramides per mouse, all liposome formulations injected i.v. were nontoxic.
6976 Langmuir, Vol. 24, No. 13, 2008
Figure 7. Therapeutic efficacy of the nSSL containing ceramides. The graph represents the percent survival of BALB/c mice inoculated i.p. with 1 × 106 C-26 colon carcinoma cells and treated i.v. with EPC- or HSPC-based nSSL. Treatment began on day 3 after tumor cell inoculation and was repeated twice for a total of three injections, 3 days apart. Untreated (triangles, black line); treated with nSSL lacking ceramides (triangles, dashed grey line); treated with C6Cer nSSL based on EPC (circles, dashed grey line); treated with C6Cer nSSL based on HSPC (circles, black line); and treated with C4Cer nSSL based on EPC (squares, dashed grey line).
3.13. Therapeutic Efficacy of Nanoliposomes Containing Ceramides in Tumor-Bearing Mice. C6Cer showed a high level of uptake into the tumor cells and was found to be very efficacious in the induction of cell death. Also, good serum stability was demonstrated for the nanoliposomes containing C6Cer. LUVs based on either EPC or HSPC (6 µmol/mouse) and containing C6Cer or C4Cer (2 µmol/mouse), with or without 2kPEG-DSPE, were evaluated for antitumor efficacy using the C-26 colon carcinoma-bearing mouse model. The control groups included mice injected with nSSL-LUV (pegylated LUV) lacking ceramide and untreated mice. Therapeutic efficacy was determined as increase in life span (ILS). The survival curves of mice inoculated with C-26 colon carcinoma cells i.p. and treated i.v. with nSSLs are presented in Figure 7. It was found that only mice treated with nSSLs containing C6Cer or C4Cer showed an increase in life span. Survival of mice treated with nSSLs containing C6Cer based on EPC or HSPC was 19 or 18 days, respectively, which corresponds to 36.7% ILS (p < 0.001) and to 28.6% ILS (p < 0.0045) compared to 13-day survival in the case of the two control groups (Figure 7). On the other hand, a statistically nonsignificant difference (p < 0.064) between survival of mice treated with non-SSL LUVs based on EPC (15 days) and untreated mice (13 days) was demonstrated (data not shown).
4. Discussion Our results indicated that the use of dispersions of ceramides alone, prepared by mixing a solution of a ceramide in organic solvents such as ethanol or ethanol/dodecane (98:2)44,45 with a buffer and/or serum results in a large increase in particle size and heterogeneity of the dispersion. This may explain their high toxicity after i.v. injection into mice. To overcome this phenomenon, and to improve pharmacokinetics and biodistribution in order to achieve significant therapeutic benefit, we developed PEGylated nanoliposomes (nSSLs) as a ceramide delivery system. This study includes an in-depth characterization of the relevant physicochemical properties of ceramides and of ceramide-containing liposomes and correlates them with their toxicity, pharmacokinetics, and therapeutic performance. 4.1. Relevant Physicochemical Properties of the Ceramides and Other Liposome Components. We characterized the physicochemical properties of ceramides, liposome-forming
KhazanoV et al.
phosphatidylcholines (EPC and HSPC), and the 2kPEG-DSPE lipopolymer. Table 1 shows the distinct differences between the partition coefficients of the different ceramides in the Dole apolar/polar two-phase system and how they compared to the two PCs and 2kPEG-DSPE. Table 1 demonstrates that the longer the N-acyl chain, the more ceramide distributes into the apolar phase. C6Cer is the shortest Cer to prefer the apolar phase (∼63%). Regarding the liposome-forming PCs, we found that the Kp of EPC is significantly smaller than that of HSPC (Table 1). The differences in the physicochemical properties of EPC and HSPC can be explained by the differences in the hydrophobic surface area, which are reduced by the presence of the double bond in the EPC molecule. C2Cer and, to a lesser degree, C4Cer showed instability in the air/water interface, while all other ceramides studied formed stable monolayers. This also agrees with C2Cer and C4Cer having the highest polarity of all ceramides used (Table 1). When the surface area per molecule of ceramides was determined at a surface pressure of 20 mN/m2, C6Cer had the largest area (50 ( 2 A2) per molecule (Table 2), reflecting the large chain mismatch between the long sphingoid base chain and the much shorter N-acyl chain (see structural model). Our data on C16Cer monolayers are in good agreement with previous studies done by Carrer and Maggio.46 The fact that the C16Cer has a smaller area per molecule than the short- and medium-N-acyl Cer is a result of its tighter packing, due to much smaller chain mismatch, which is large for the short- and medium-N-acyl Cer. The CAC results (Table 2) have two important implications. First, they demonstrate a very large difference (>104-fold) between both C2Cer and C6Cer compared to C16Cer, while the difference between C2 and C6 ceramides is relatively small (only 2-fold). This again reflects the impact of the large chain mismatch that exists in C2Cer and C6Cer, which may result in interdigitation and therefore a smaller hydrophobic surface area exposed to the aqueous phase. Second, at concentrations above 1 × 10-4 M, the level of monomers in the aqueous phase for C6Cer, and even more for C2Cer, will be much higher than that for C16Cer. This should have a large impact on the rate at which the various ceramides are transferred between the lipid membranes and the aqueous phase. Indeed, we demonstrate that higher CAC leads to higher transfer rate to cells in culture. This is in agreement with a study done by Sot et al.,47 which showed that both C2Cer and C6Cer behave as typical “soluble amphiphiles” with a CAC in the 5 µM range. Their values are lower than the CAC values measured by us. This discrepancy is explained by the different methods used for CAC determination; Sot et al. used an assay that requires addition of the fluorophore ANS, which becomes a part of the assembly and therefore affects it and may shift the CAC downward.37 We used a surface tension measurement, which does not require a probe and therefore introduces fewer artifacts than other methods.32,48 4.2. Packing Parameter of Ceramides and of Liposomal Formulations. Packing parameter is used routinely to classify amphiphiles and to predict the amphiphile assembly type and structure.49 For a description of structure–composition relationships in a multicomponent (amphiphile) system, the “additive packing parameter” (APP) or “effective additive packing parameter” was used.24 (46) Carrer, D. C.; Maggio, B. Biochim. Biophys. Acta 2001, 1514, 87–99. (47) Sot, J.; Goni, F. M.; Alonso, A. Biochim. Biophys. Acta 2005, 1711, 12–19. (48) Mukherjee, P.; Musels, K. J., Critical Micelle Concentrations of Aqueous Surfactant Systems. NSRDS-NBS36; National Bureau of Standards: Washington, D.C., 1971; pp 8-18. (49) Israelachvili, J. N.; Mitchel, D. J.; Ninham, B. W. J. Chem. Soc., Faraday Trans. II 1976, 72, 1525–1525.
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Table 8. Calculation of Packing Parameter of Ceramides
lipid
Vj, partial specific volume (mL/g)
V, volume of the hydrophobic tail (nm3)
L, length of the hydrophobic tail (nm)
A, area of the hydrophilic headgroup (nm2)
PP, packing parameter
C2Cer C6Cer C16Cer
1.071 1.122 1.017
0.590 0.724 0.891
1.10 1.30 1.80
0.39 0.37 0.39
1.38 1.51 1.27
Length of the hydrophobic tail (acyl chain), L, was calculated according to the work of Israelachvili et al.49 published on the basis of X-ray data: L ) 0.08 (1.5 + 1.265n - 0.9ndb), where n is the average number of carbons in the two acyl chains and ndb is the number of double bonds. The area of the hydrophilic headgroup of C16Cer is from Holopainen et al.,50 and of those of C2 and C6Cer are estimated from Huang et al.51
Table 9. Calculation of Packing Parameter of C6Cer from Maximum Loading Capacity into PC Bilayers EPC a
no.
14 17 15 18 average a
Figure 8. Influence of the APP of the PC-C6Cer formulations on the physical stability of the liposomal formulations. Note that extrapolation to zero stability gives the value, which is very close to 1.0, of the upper limit for the formation of stable liposomes.
Our calculations of PP of C2Cer, C6Cer, and C16Cer are based on the measurements of the partial specific volume of EPC/Cer in liposomes. In all liposomal formulations, the mole ratio of 0.77:0.23 was used. From these values plus the partial specific volume of pure EPC, based on the additivity of volumes, the partial specific volume and volume of the hydrophobic tail of ceramides were calculated (Table 8). The length of the hydrophobic tail was calculated as the average value of the two asymmetric hydrocarbon chains by following the procedure of Israelachvili et al.49 The area of the hydrophilic headgroup of the C16Cer was taken from Holopainen et al.50 We took into account the fact that C6Cer slightly decreases the headgroup area, while C2Cer does not induce any significant effect on headgroup area.51 The value of PP of the long-chain C16Cer calculated in Table 8 is in good agreement with the literature,46 where, for longchain ceramides, PP values of 1.22 to 1.27 were obtained, depending on the variations of surface pressure. No such data are available for ceramides having shorter N-acyl chains. The calculated PP value of 1.51 for C6Cer came from our partial specific volume measurements and geometrical parameters of the ceramides and are in agreement with our monolayer results and physical stability follow-up study of liposomal formulations composed of PCs and C6Cer. For the formation of liposomes, the APP of the liposome bilayer components has to be within the range of 0.74-1.0.19 Calculations of the APP of formulations 1, 2, 13, and 16 (Table 3) composed of C6Cer and/or PC lipid mixtures give high APP values, and extrapolation to zero stability (i.e., unstable liposome formulations) gives a value that is very close to the upper limit of the APP for liposome formulations (∼1.0, Figure 8). Supporting the high calculated value of the C6Cer PP is the determination of the (50) Holopainen, J. M.; Brockman, H. L.; Brown, R. E.; Kinnunen, P. K. J. Biophys. J. 2001, 80, 765–775. (51) Huang, H.; Goldberg, E. M.; Zidovetzki, R. Eur. Biophys. J. 1998, 27, 361–366.
HSPC
2k
C6Cer
PEG-DSPE
fraction PP fraction PP fraction PP fraction 0.575 0 0.54 0
0.80 0.80 0.80 0.80
0 0.575 0 0.54
0.82 0.82 0.82 0.82
0.075 0.075 0.11 0.11
0.89 0.89 0.78 0.78
0.35 0.35 0.35 0.35
PP 1.505 1.538 1.479 1.510 1.51 ( 0.02
Numbers (no.) were taken from Table 3.
loading capacity of C6Cer into PC bilayers. It was determined that liposomal formulations containing 7.5 as well as 11 mol % 2kPEG-DSPE have the maximal loading capacity of C Cer into 6 PL bilayers of about 35 mol %. Above this value, C6Cer separates from the liposome, as an aggregate and precipitate. At 35 mol % of C6Cer, the APP value of the three-component formulation containing liposome-forming PC and 7.5 or 11 mol % 2kPEGDSPE is as high as 1.0.19 The PP values of all the components of the lipid bilayers are presented in Table 9. For 2kPEG-DSPE the value of “effective” PP was used.24 The value of the PP of C6Cer calculated from the maximum loading capacity into EPC and HSPC bilayers is 1.51 ( 0.02 (Table 9). The APP of all other ceramides and liposomal formulations varying in mole % of liposome-forming lipids and 2kPEG-DSPE are presented in Table 3. When APP was calculated for liposomal formulations based on either EPC or HSPC without ceramides and varying in mole % of liposome-forming lipids and lipopolymer (Nos. 1-8), we found that they fell in the range of 0.77-0.82. As expected, the addition of ceramides increases the PP (to 0.89-1.0). Our results (Figure 9) show a good correlation between the values of PP of ceramides with the partial specific compressibility of the various PC-Cer liposomes. The significant increase of compressibility with the addition of 23 mol % of ceramides (see Figure 4) can be explained by the formation of highly compressible cavities (free volume) and is related to chain mismatch, which is greatest in C6Cer. The volume of the ceramide-induced cavities is correlated with the PP of ceramides and plays an important role in modulating their apoptotic activity. 4.3. Stability of Ceramide-PC Liposomes. On the basis of consideration of the APP,19 we postulated that the addition of 2kPEG-DSPE, by increasing the miscibility of the ceramides with the other liposome membrane components, as well as reducing membrane cavity formation can improve the stability of ceramideenriched liposomes. Our results confirmed this. We demonstrated that increasing the mole % of the 2kPEG-DSPE lipopolymer increases the ceramide level in the LUV membrane for C6Cer (see Nos. 13, 14, and 15 in Table 3). Regarding the liposome stability, we showed that LUVs containing ceramide and lacking 2kPEG-DSPE were unstable and disintegrated over a short time (from 1.25 to 3.5 months, Table 3), but liposomal formulations containing 2kPEG-DSPE were stable for 4 to 24 months, depending on ceramide chain length (Table 3). This was correlated with the
6978 Langmuir, Vol. 24, No. 13, 2008
Figure 9. Correlation between the PP of ceramides and the compressibility of PC-Cer bilayers. Partial specific compressibility values, as well as specific volumes, can be considered to be the sum of three main j ) Kintr + Kcav - ∆Khydr, where Kintr is the intrinsic components: K compressibility of the lipid bilayer determined by the van der Waals dimensions and packing density of lipid molecules, Kcav is the compressibility of the cavities (packing defects), and ∆Khydr is the compressibility changes caused by headgroup hydration, which makes a negative contribution to the total compressibility. Note that Kintr and ∆Khydr for all ceramides are similar whereas the Kcav component is different.
APP of the liposomes; lower APP (∼0.8) gave better stability (Table 3). Moreover, when 37 mol % of cholesterol was included in the liposomes containing C6Cer, APP g1.0, and the entire lipid bilayer was in the liquid ordered (LO) phase, their stability was rather poor, and these liposomes disintegrated within ∼7 days (No. 21 in Table 3). Our results may be related to the findings of London and London25 that ceramide competed with cholesterol, as the addition of 9 mol % ceramide to the bilayer lipids displaced 50% of the total cholesterol. They explain it by the formation of “rafts”. Similar results were obtained by Sot et al.47 and others.52,54–56 Nybond et al.53 suggested that only ceramides having an N-acyl chain of at least eight carbons induce such displacement. Sot et al. also demonstrated that the binary mixtures of C16 (but not of C2 or C6Cer) with sphingomyelin form Cer-enriched detergent-resistant gel domains.57 In our studies, liposomes composed of PC and C2Cer or C6Cer were less stable than liposomes containing medium C8Cer or C16Cer. The difference in the behavior of the different ceramides used is probably related to the degree of their hydrocarbon chain mismatch. C2Cer and especially C6Cer have pronounced chain mismatch (see structural model), which introduces a large free volume into the lipid bilayer or, alternatively, interdigitation (see structural model and refs 55 and 56). Regarding the liposomeforming PC, we found that liposomes in which the high-Tm HSPC is the liposome-forming PC were somewhat less stable than liposomes based on the low-Tm EPC. This may be related to their smaller PP and to the presence of the cis double bonds in one of the EPC acyl chains, which allows the molecule to be more flexible and to better fill the free volume introduced to the liposome bilayer by the ceramide. 4.4. ThermotropicBehaviorofHSPC-ceramideLiposomes. The thermotropic behavior studied by DST (Figures 1and 2) and (52) Alanko, S. M.; Halling, K. K.; Maunula, S.; Ramstedt, B. Biochim. Biophys. Acta 2005, 1715, 111–121. (53) Nybond, S.; Bjorkqvist, Y. J.; Ramstedt, B.; Slotte, J. P. Biochim. Biophys. Acta 2005, 10, 61–66. (54) Chiantia, S.; Kahya, N.; Ries, J.; Schwille, P. Biophys. J. 2006, 90, 4500– 4508. (55) Huang, C.; Mason, J. T. Biochim. Biophys. Acta 1986, 864, 423–470. (56) Mason, J. T.; Cunningham, R. E.; O’Leary, T. J. Biochim. Biophys. Acta 1995, 1236, 65–72. (57) Sot, J.; Bagatolli, L. A.; Gon˜i, F. M.; Alonso, A. Biophys. J. 2006, 90, 903–914.
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DSC (Figure 3) demonstrated that HSPC-ceramide miscibility in the liposome bilayer is greatly affected by ceramide N-acyl chain length and by the presence of 2kPEG-DSPE. The most extensive immiscibility (expressed as phase separation) occurred for an HSPC mixture with C6Cer, while C2Cer had a smaller effect, and C16Cer showed good miscibility (compare panels A, B, and C in Figure 1). Liposome phase separation introduced by C6Cer is explained by the difference in temperature range of the SO to LD phase transition of the membrane components and by the degree of acyl chain mismatch.58 Such chain mismatch introduces more free volume in the bilayer and, combined with the small ceramide headgroup, is the reason that C6Cer has the highest PP (see Table 3 and the structural model). This is also correlated with the values of surface area per molecule at the air/water interface (Table 2). Mixing of lipids with matched and mismatched chains leads to phase separation, as was previously demonstrated for mixing dimyristoylphosphatidylcholine (DMPC) and C24-sphingomyelin.58 C16Cer has the opposite effect on the HSPC thermotropic behavior; no phase separation was observed and the Tm of the lipid assemblies increases. Elevation of Tm without much broadening of the peak implies miscibility and cooperative interaction between HSPC and C16Cer (even at 75 mol % of C16Cer), although the assemblies formed may be heterogeneous, and not all are liposomes. Although the headgroup of C16Cer has the same size as that of C2Cer and C6Cer, C16Cer lacks chain mismatch (see structural model). So far, most of the studies done on the thermotropic behavior of ceramide/PL mixtures were done using long-chain ceramides. It was previously shown that the phase behavior of C16Cer/DMPC bilayers is highly complex.59 Increasing the mole fraction of ceramide caused broadening of phase transition, a rise in Tm, and phase separation. Unlike in our studies with HSPC/C16Cer mixtures, the phase transition diagrams showed phase separation between DMPCand C16Cer-rich domains.59 In our three-component system (HSPC, C6Cer, and 2kPEG-DSPE), Tm increases as well, but there is no phase separation. This difference may be due to the presence of the 2kPEG-DSPE in our lipid assemblies, which further improves the packing of the lipid components in the bilayer, thereby increasing miscibility and eliminating phase separation. We demonstrated that indeed, the 2kPEG-DSPE, which has a small PP due to its large and highly hydrated headgroup (see structural model) improves the miscibility of liposome components. Figure 2A,B shows that the addition of 7.5 mol % 2kPEGDSPE to the LUVs containing C6Cer abolished the phase separation completely. This correlates with our results on liposome membrane compressibility. The addition of 7.5 mol % 2kPEGDSPE to either EPC or HSPC liposomes decreased the partial specific compressibility of the lipid bilayers, indicating tighter packing. A decrease in compressibility also reflects dehydration of the lipid bilayer,43 which plays an important role in the stabilization of the PEG-modified liposomes. Tight packing of the lipids in the liposome bilayer results in increased stability of the liposomal formulations (see Table 3). We demonstrated that, by adding 2kPEG-DSPE, the desorption rate of the ceramides from the liposomes was slowed down, and liposomal stability was improved. Our working hypothesis, based on the formulation of stable liposomes using the APP concept, was proven. 4.5. Relationship between Cytotoxic Activity of Ceramides and Their Cellular Uptake. Our main goal was to develop stable liposomes, containing >10 mol % ceramides, that show (58) Bar, L. K.; Barenholz, Y.; Thompson, T.E. Biochemistry 1997, 36, 2507– 2516. (59) Holopainen, J. M.; Lemmich, J.; Richter, F. Biophys. J. 2000, 78, 2459– 2469.
Characterization of Ceramide-Containing Liposomes
therapeutic activity against tumors in animals. Formulations composed either of EPC or HSPC as the liposome-forming lipid were cytotoxic against C-26 and OV-1063 cancer cell lines (Table 5). According to our results, C6Cer is the most cytotoxic of all ceramides studied, and C16Cer is almost devoid of cytotoxicity, in good agreement with the results of Shabbits and Mayer.60 The cytotoxic effects of nSSL-ceramide formulations required longer incubation times compared to “free” ceramides and to ceramides in conventional, non-SSL nanoliposomes, which correlated with the uptake of ceramides by the cells (Table 6). The cytotoxic activity of ceramide ethanolic dispersions (free ceramides) was comparable to that shown by other researchers.3 Differences in cytotoxicity may also be related to the CAC and state of aggregation of various ceramides in aqueous medium, as these may affect their cell uptake. The results presented in Table 2 show that C2Cer and C6Cer have a relatively high CAC, in the range of 10-5M. The CAC of C16Cer was 105-fold lower. Ceramide uptake is related to their CAC, as in the concentrations used by us in the cytotoxicity studies in vitro, C2Cer and C6Cer exist mostly as monomers/dimers, and C16Cer exists as an assembly (of yet unknown nature). The level of ceramide cell uptake explains the differences in cytotoxic activity between short-, medium-, and long-chain ceramides. Free and liposomal 14C6Cer were efficiently taken up and metabolized by C-26 cells in a time-dependent manner, while free 14C16Cer and liposomal 14C16Cer were released and taken up by cells to a much lower extent than 14C6Cer (Table 6). These results are consistent with those of Venkataraman and Futerman,61 which showed that the uptake of free long-chain C16Cer and its metabolism to SPM are much lower than that of medium-chain C6Cer. They suggest that the difference is due to a very slow transfer rate of long-chain ceramides into biological membranes. C6Cer present in nSSLs is transferred more slowly to the cells than either free C6Cer or C6Cer in non-SSL nanoliposomes, and, therefore, the lipopolymer allows sustained and slow release of ceramide from the liposomes (Table 5). The slower release of C6Cer from liposomes, especially from nSSLs, may explain the differences in time-dependent C6Cer biodistribution and in cytotoxic activity between free and nSSL ceramide, particularly for short time periods. At all times tested, it appears that liposomal C6Cer was taken up by the cells independently of the liposomal 3H-DPPC (Table 6). This suggests that C6Cer is taken up by cells by itself, without the liposome-forming PC, either after being released from the lipid assemblies or by diffusion from the lipid assemblies during their collision with the cells. The PC of the lipid assemblies is taken up by the cells at a much slower rate than the C6Cer, either by transfer between liposomes and cells or by the small uptake of intact liposomes by the cells. Both mechanisms were demonstrated in other cells in cultures in the past for PC.62 Once taken up by the cells, C6Cer and C16Cer are metabolized, by well-established pathways, mainly to C6SPM or C16SPM and, to a lesser extent, C6GlcCer.63,64 Since ceramides have a small headgroup, they can “flip-flop” from the external to the internal plasma membrane leaflet much faster than can phospholipids, which have a much larger headgroup,65 acting like an energetic barrier that slows down phospholipid movement through the hydrophobic region of the lipid bilayer. Also, chain mismatch (60) Shabbits, J. A.; Mayer, L. D. Biochim. Biophys. Acta 2003, 1612, 98–106. (61) Venkataraman, K.; Futerman, A. H. Biochim. Biophys. Acta 2001, 1530, 219–226. (62) Yechiel, E.; Barenholz, Y.; Henis, Y. I. J. Biol. Chem. 1985, 260, 9123– 9131. (63) Bai, J.; Pagano, R. E. Biochemistry 1997, 36, 8840–8848. (64) Rosenwald, A. D.; Pagano, R. E. AdV. Lipid. Res. 1993, 26, 101–118. (65) Goni, F. M.; Alonso, A. Biochim. Biophys. Acta 2006, 1758, 1902–1921.
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of short- and medium-chain ceramides should further facilitate the flip-flop movements as a result of packing defects and increased free volume in the lipid bilayer. This may explain why C6Cer is taken up by cells and, in contrast to long-chain ceramides, is readily accessible to glucosylceramide synthase (GCS), which methabolizes ceramide to C6GlcCer. As a result of the normal SPM trafficking, medium-chain SPM reaches the plasma membrane and, being much more polar than the long-chain cellular SPM, is desorbed to the growth medium and associates with FCS lipoproteins and proteins. This explains why more of the 14C6 SPM relative to the long-chain 14C16 SPM is present in the cell growth medium (Figure 6A,B). We demonstrated that cancer cells in culture, when treated with short- or medium-chain ceramides delivered either free or as part of a liposomal formulation, undergo apoptosis. Distinct features of apoptosis were shown by several methods (see Results and Table 7) and are in agreement with previously published work.3,6,33 Induction of apoptosis by long-chain C16Cer was 3 times lower than that by C6 or C2Cer, which we explained by its lower uptake into cells (Table 6), and this results in lower cytotoxicity (Table 5). It is well-known that liposomes may aggregate in the presence of serum.66 Large aggregates may clog blood vessels and are directed to the lungs and reticuloendothelial system (RES). Such effects reduce the benefits of liposome delivery and partially explain liposome toxicity. In vivo, such aggregation prevents passive targeting of liposomes through the fenestrated blood vessels of tumor tissues. We followed the stability of different liposomal LUV formulations containing C2Cer, C6Cer, or C16Cer in the presence or absence of FCS by two independent methods: DLS and turbidity measurements (Table 4). Serum did not affect the size of nanoliposomes lacking ceramides or those that include C2Cer or C6Cer. However, those containing C16Cer increase their size to a large extent (probably due to aggregation) in the presence of FCS. 4.6. Toxicity and Therapeutic Efficacy of the CeramidePC Liposomes. Toxicity studies performed in mice and also in dogs (data not shown) revealed that the various liposomal ceramide formulations that were stable in serum were also nontoxic upon i.v. administration. When we evaluated the therapeutic efficacy of liposomes containing C6Cer in mice, we found that only nSSLs containing C6Cer showed therapeutic activity, while non-SSL-C6Cer lacked therapeutic efficacy. Also, there was a correlation between pharmacokinetics and therapeutic efficacy of C6Cer. About 2% of the total C6Cer injected as SSL-C6Cer was found in tumor tissue, compared to barely detectectable levels of C6Cer delivered by non-SSL-C6Cer (data not shown). This ceramide biodistribution suggests that the therapeutic activity of liposomes containing ceramides is due to their passive targeting to tumors, as was suggested before for Doxil.26 This passive targeting of the nanoliposomes occurs as a result of their extravasation through the fenestrated endothelium of the tumor blood vessels, which increase in primary and metastatic tumors.67 The superior activity of nSSLs containing C6Cer over non-SSL formulations appears to be due to the presence of the lipopolymer 2kPEG-DSPE, which acts as a steric barrier, reducing uptake by the RES and prolonging circulation time.68 To the best of our knowledge, the i.v. injection of the sterically stabilized liposomal C6Cer by us was one of the first trials to explore the feasibility and efficacy of ceramide application in (66) Jones, M. N.; Nicholas, A. R. Biochim. Biophys. Acta 1991, 1065, 145– 152. (67) Folkman, J. AdV. Cancer Res. 1985, 43, 175–203. (68) Gabizon, A.; Martin, F. Drugs 1997, 54, 15–21.
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antitumor therapy.69 Currently there are several groups working on the development of liposomal ceramide formulations. The group of Shabbits and Mayer showed that i.v. treatment of mice having J774 ascites tumor by cationic liposomes containing C16Cer resulted in a 25.8% ILS over control liposomes.70 Stover and Kester71 were able to include only up to 10 mol % of ceramide into the liposomes consisting of DOPC/DOPE/Chol/450PEGC8Cer/C6Cer (40:30:10:10:10). On the basis of physicochemical characterization of these liposomes, they should have two disadvantages, both related to loss of steric stabilization: first, the mismatched short-chain 450PEG-C8Cer will not supply sufficient steric stabilization because of its small (450 Da) PEG moiety;72 second, the 450PEG-C8Cer, being a medium-chain ceramide, will desorb relatively quickly from the liposomes.72 Additionally, on the basis of the concept of APP as a way to predict the stability of liposomes (Table 3) and confirmation by London and London,25 we demonstrated that the stability of nSSLs containing both cholesterol and C6Cer is low. In summary, in this study we demonstrated that, by controlling the APP of the liposomes through the inclusion of the lipopolymer 2kPEG-DSPE, we were able to develop nanoliposomes containing various ceramides (nSSL-Cer) that were stable during storage at 4 °C, as well as in biological fluids such as plasma. Ceramides in these nSSLs preserve their cytotoxic activity. Cytotoxicity strongly depends on ceramide N-acyl chain length, which affects the degree of cell uptake. The inclusion of the 2kPEG-DSPE into nanoliposomes containing ceramides (and especially of C6Cer) slowed down ceramide uptake by cells, suggesting that the lipopolymer decreases the desorption rate of ceramides from the liposomes, as was also expressed by an increase in the level of C6Cer reaching the tumor, which explains the superior therapeutic efficacy of nSSLs containing C6Cer. Our ongoing research is aimed at evaluating the possibility of including ceramides into nSSLs containing chemotherapeutic agents and the potential of such combined therapy. Acknowledgment. The help of Prof. Peter J. Slotte, Abo (69) Barenholz, Y.; Khazanov, E.; Schillemans, J. International Patent WO 2004087097, 2004. (70) Shabbits, J. A.; Mayer, L. D. Anticancer Res. 2003, 23, 3663–3669. (71) Stover, T.; Kester, M. J. Pharmacol. Exp. Ther. 2003, 307, 468–475. (72) Woodle, M. C.; Lasic, D. D. Biochim. Biophys. Acta 1992, 1113, 171– 199.
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Academi University, Turku, Finland, in the monolayer measurements is acknowledged with great pleasure. Mr. Sigmund Geller is acknowledged for help in editing the manuscript. This work was supported in part by the Barenholz Fund.
Glossary APP CBS Cer CAC CLSM DLS DSC DST EPC FCS GlcCer HSPC ILS LUV MB MLV ODN PC 2kPEGDSPE PL PP SPM SSL nSSL TLC Tm TR
AbbreViations additive packing parameter citrate buffered saline ceramide critical aggregation concentration confocal laser scanning microscope dynamic light-scattering differential scanning calorimetry differential scanning turbidity egg phosphatidylcholine, fetal calf serum glucosylcerebrosides hydrogenated soy phophatidylcholine increase in life span large unilamellar liposome methylene blue multilamellar liposome oligodeoxynucleotide phosphatidylcholine N-carbamyl-poly-(ethylene glycol methyl ether)-1,2distearoyl-sn-glycero, 3-phosphoethanolamine triethyl ammonium salt phospholipid packing parameter sphingomyelin sterically stabilized liposome sterically stabilized nanoliposome thin layer chromatography temperature at which the maximum change in heat capacity of main gel to liquid crystalline phase transition occurs ratio of turbidities determined at two different wavelength
LA800207Z
