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Interaction of Hydrated r-Galactosylceramide with Nonionic Surfactants and Formation of Dispersions Including Gel Phases Minoru Nakano,† Yousuke Nakatani,† Atsuhiko Sugita,† Tomoari Kamo,† Takenori Natori,‡ and Tetsurou Handa*,† Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan, and Pharmaceutical Development Laboratory, Kirin Brewery Company, Limited, 1-2-2 Souja-Machi Maebashi, Gunma 371-0853, Japan Received January 14, 2003. In Final Form: February 28, 2003 Hydrated R-Galactosylceramide (R-GalCer) has a high gel to liquid crystalline phase transition temperature and forms a gel phase at room temperature, which separates from an excess aqueous medium. In this study, R-GalCer was dispersed into the aqueous medium by high-pressure emulsification with nonionic surfactants. Using Pluronic, stable dispersions with a hydrodynamic diameter of ca. 200 nm were obtained. Small-angle and wide-angle X-ray diffraction from the dispersion showed patterns similar to those of the hydrated R-GalCer indicating the formation of the gel-containing nanoparticles. Differential scanning calorimetry (DSC) also revealed the existence of gel phases in the dispersion. However, the particles aggregated when Tween was used as a stabilizer. On the other hand, the solubility of R-GalCer was higher in Tween micelles than Pluronic micelles. DSC results suggested that Pluronic was less compatible with the R-GalCer gel phase than Tween, which was related to the stability of the dispersions and solubility of R-GalCer in micelles.
Introduction R-Galactosylceramide (R-GalCer, Figure 1a) is a glycosylceramide containing R-anomeric glucose with a long fatty acyl chain (C26) and a phytosphingosine base. This lipid was identified as a ligand for the invariant T cell antigen receptor expressed by natural killer T (NKT) lymphocytes.1-3 NKT cell recognition of R-GalCer bound to the monomorphic CD1d molecule triggers proliferation, cytokine release, and cytotoxic activity.4-6 R-GalCer has a potent antitumor activity and may be a useful agent for cancer therapy.7,8 In addition, R-GalCer prevents development of autoimmune diabetes in nonobese diabetic mice, suggesting that R-GalCer may be useful for the treatment of human diseases characterized by Th1-mediated pathology.9 Although this glycosphingolipid has large potentiality for therapeutic intervention, its significant low solubility * Corresponding author. E-mail:
[email protected]. † Kyoto University. ‡ Kirin Brewery Co., Ltd. (1) Bendelac, A.; Rivera, M. N.; Park, S. H.; Roark, J. H. Annu. Rev. Immunol. 1997, 15, 535-562. (2) Godfrey, D. I.; Hammond, K. J. L.; Poulton, L. D.; Smyth, M. J.; Baxter, A. G. Immunol. Today 2000, 21, 573-583. (3) Smyth, M. J.; Crowe, N. Y.; Hayakawa, Y.; Takeda, K.; Yagita, H.; Godfrey, D. I. Curr. Opin. Immunol. 2002, 14, 165-171. (4) Kawano, T.; Cui, J.; Koezuka, Y.; Toura, I.; Kaneko, Y.; Motoki, K.; Ueno, H.; Nakagawa, R.; Sato, H.; Kondo, E.; Koseki, H.; Taniguchi, M. Science 1997, 278, 1626-1629. (5) Spada, F. M.; Koezuka, Y.; Porcelli, S. A. J. Exp. Med. 1998, 188, 1529-1534. (6) Burdin, N.; Brossay, L.; Koezuka, Y.; Smiley, S. T.; Grusby, M. J.; Gui, M.; Taniguchi, M.; Hayakawa, K.; Kronenberg, M. J. Immunol. 1998, 161, 3271-3281. (7) Morita, M.; Motoki, K.; Akimoto, K.; Natori, T.; Sakai, T.; Sawa, E.; Yamaji, K.; Koezuka, Y.; Kobayashi, E.; Fukushima, H. J. Med. Chem. 1995, 38, 2176-2187. (8) Kobayashi, E.; Motoki, K.; Uchida, T.; Fukushima, H.; Koezuka, Y. Oncol. Res. 1995, 7, 529-534. (9) Hong, S.; Wilson, M. T.; Serizawa, I.; Wu, L.; Singh, N.; Naidenko, O. V.; Miura, T.; Haba, T.; Scherer, D. C.; Wei, J.; Kronenberg, M.; Koezuka, Y.; Kaer, L. V. Nat. Med. 2001, 7, 1052-1062.
Figure 1. Molecular structures of (a) R-Galactosylceramide, (b) Pluronic, and (c) Tween surfactants.
into water and other solvents hinders its clinical administration. For in vitro and in vivo experiments, R-GalCer is normally administrated either as a dimethyl sulfoxide solution or by solubilizing into Polysorbate20 (Tween80, Figure 1c) micelles, respectively. Of course, the reduced use of these solubilizing materials is clinically desirable. One promising way is liposome administration. However, R-GalCer cannot form liposomes alone, presumably due to its being in a gel state at room temperature and its insufficient hydrophilicity. Although its incorporation into phospholipid liposome bilayers is possible, it is limited by the low miscibility with phospholipids.10 In general, glycosylceramides and phospholipids are completely miscible in the liquid crystalline state but poorly miscible in the gel state.11,12 Since glycosphingolipids in nature have a high gel-liquid crystal transition temperature and a (10) Nakano, M.; Inoue, R.; Koda, M.; Baba, T.; Matsuhaga, H.; Natori, T.; Handa, T. Langmuir 2000, 16, 7156-7161. (11) Ruocco, M. J.; Shipley, G. G.; Oldfield, E. Biophys. J. 1983, 43, 91-101.
10.1021/la0340605 CCC: $25.00 © 2003 American Chemical Society Published on Web 04/23/2003
Formation of R-GalCer Dispersions
capacity to form an extensive hydrogen-bonding network, they can form detergent-insoluble gel-phase domains.13-16 An alternative method is to disperse the glycolipids in the presence of stabilizing surfactants. Nanosuspensions or solid lipid nanoparticles have been widely investigated during recent years.17,18 These are colloidal particles composed of the drug or solid lipids, respectively, and stabilized by an emulsifier. It is suggested that these nanoparticles can improve the bioavailability and dose proportionality of poorly soluble drugs. To prepare such nanoparticles, Pluronic and Tween surfactants are often used as stabilizers.17 Pluronic (Figure 1b) is an amphiphilic triblock copolymer and is widely applied to drug delivery systems.19-21 In addition, Pluronic has attracted increased attention because it was discovered that Pluronic interacts with multidrug-resistant cancer tumors resulting in marked sensitization of these tumors with respect to various anticancer agents.21,22 We previously demonstrated that cubic and inverted hexagonal phases can be dispersed to form cubosomes and hexosomes using Pluronic F127 as an emulsifier, which absorbs at the particle surface and does not disturb the internal liquid crystalline phases.23,24 Thus, it is suggested that Pluronic can also be an effective emulsifier of the glycolipid’s gel phase that shows little miscibility with the surfactant. In the present study, the differences between two different types of surfactants, Pluronic and Tween, in the solubility of R-GalCer into the micelles and the stability of the dispersions including the gel phases are discussed with relevance to the miscibility with the glycolipid. Experimental Section Materials. R-GalCer (KRN7000) was synthesized as described previously.25 The purity (>98%) was determined by thin-layer chromatography. Pluronic F127 (PEO99-PPO67-PEO99), L64 (PEO13-PPO29-PEO13), and P84 (PEO19-PPO43-PEO19) were provided by BASF Japan Ltd. (Osaka, Japan). Pluronic F68 (PEO76-PPO29-PEO76), F88 (PEO103-PPO39-PEO103), and F108 (PEO127-PPO48-PEO127) were provided by Asahi Denka Co., Ltd. (Tokyo, Japan). Tween20 (polyoxyethylenesorbitan monolaurate) and Tween80 (polyoxyethylenesorbitan monooleate) were purchased from Sigma (Osaka). 1,6-Diphenylhexatriene (DPH) was purchased from Molecular Probes Inc. (Eugene, OR). These were used without further purification. Other materials described later without notation were purchased from Wako Pure Chemical Industries Ltd. (Osaka). (12) Gardam, M.; Silvius, J. R. Biochim. Biophys. Acta 1989, 980, 319-325. (13) Schroeder, R.; London, E.; Brown, D. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 12130-12134. (14) Brown, R. E. J. Cell Sci. 1998, 111, 1-9. (15) Masserini, M.; Ravasi, D. Biochim. Biophys. Acta 2001, 1532, 149-161. (16) Ramstedt, B.; Slotte, J. P. FEBS Lett. 2002, 531, 33-37. (17) Mehnert, W.; Ma¨der, K. Adv. Drug Delivery Rev. 2001, 47, 165196. (18) Mu¨ller, R. H.; Jacobs, C.; Kayser, O. Adv. Drug Delivery Rev. 2001, 47, 3-19. (19) Guzman, M.; Garcia, F. F.; Molpeceres, J.; Aberturas, M. R. Int. J. Pharm. 1992, 80, 119-127. (20) Miyazaki, S.; Tobiyama, T.; Takada, M.; Attwood, D. J. Pharm. Pharmacol. 1995, 47, 455-457. (21) Kabanov, A. V.; Batrakova, E. V.; Alakhov, V. Y. J. Controlled Release 2002, 82, 189-212. (22) Kabanov, A. V.; Batrakova, E. V.; Alakhov, V. Y. Adv. Drug Delivery Rev. 2002, 54, 759-779. (23) Nakano, M.; Sugita, A.; Matsuoka, H.; Handa, T. Langmuir 2001, 17, 3917-3922. (24) Nakano, M.; Teshigawara, T.; Sugita, A.; Leesajakul, W.; Taniguchi, A.; Kamo, T.; Matsuoka, H.; Handa, T. Langmuir 2002, 18, 9283-9288. (25) Morita, M.; Sawa, E.; Yamaji, K.; Sakai, T.; Natori, T.; Koezuka, Y.; Fukushima, H.; Akimoto, K. Biosci. Biotechnol. Biochem. 1996, 60, 288-292.
Langmuir, Vol. 19, No. 11, 2003 4605 Preparation of Dispersions. Aqueous dispersions of R-GalCer were prepared using nonionic surfactants as a stabilizer. R-GalCer (6.6 mg), Pluronic or Tween (13.2 mg), and saline (150 mM NaCl, 30 mL) were mixed and vortexed at 80 °C. Further size reduction of roughly dispersed particles was performed using a high-pressure emulsifier (nanomizer system YSNM-1500-5, Yoshidakikai Co. Ltd., Nagoya, Japan) under a pressure of 130 MPa for 30 min at 25 °C. The particle size of the dispersions was determined from dynamic light scattering (DLS) measurements (Photal LPA-3000/3100; Otsuka Electronic Co., Osaka) at 25 °C, in which the mean hydrodynamic diameter was determined by the cumulant method. The dispersions were kept at room temperature, and their size was periodically measured. The dispersions were concentrated (to greater than 10 mg/mL lipids) by ultrafiltration (Millipore) for X-ray diffraction and differential scanning calorimetry experiments. Small- and Wide-Angle X-ray Diffraction. The concentrated R-GalCer/F127 dispersion or hydrated R-GalCer was put into a thin-walled glass capillary (W. Mu¨ller, Berlin, Germany; 1.5 mm o.d., 1/100 mm wall thickness). X-ray diffraction experiments were carried out with Cu KR radiation generated by a Rigaku RU-200 rotating anode X-ray generator (Rigaku Co., Tokyo; 40 kV, 150 mA) with a graphite monochromator (wavelength, 1.5418 Å) and a pinhole collimator (diameter, 0.5 mm). The diffraction pattern was recorded with an imaging plate (Fuji Film Ltd.) in a flat camera. The camera length was 303 mm for small-angle and 150 mm for wide-angle diffraction, covering 0.013 Å-1 e s e 0.4 Å-1, where s is the scattering vector and is given by 2 sin(θ/2)/λ, θ is the diffraction angle, and λ is the X-ray wavelength, and 180- and 90-min exposures were taken for smalland wide-angle diffraction, respectively. The recorded images were read on a Rigaku imaging plate reader, and the digitized data were analyzed on a Rigaku R-AXIS IV system. For wideangle diffraction, the scattering from solvent was subtracted. Differential Scanning Calorimetry (DSC). Calorimetry experiments were performed on a DSC3200 system (Mac Science Co., Yokohama, Japan) over the temperature range between 10 and 85 °C at a heating/cooling rate of 1 °C/min. The concentrated R-GalCer/F127 dispersion was put into an aluminum cell. For measurements of hydrated samples, R-GalCer or R-GalCer/ nonionic surfactant mixtures of ca. 3 mg were hydrated above 80 °C with excess saline in the sample cell. The saline was used as a reference. Solubility Experiment. The solubility of R-GalCer in surfactant solutions was investigated using about a 10-fold higher surfactant concentration than in the dispersion. R-GalCer (2.2 mg), Pluronic or Tween (50 mg), and saline (10 mL) were mixed at 80 °C. The mixture was then cooled to room temperature. The solubility of R-GalCer was visually discriminated by checking whether there was any precipitate. Steady-State Fluorescence Anisotropy. For fluorescence experiments, three samples containing fluorescent probe DPH (1.3 µM) were prepared: The R-GalCer/F127 dispersion (0.22 mg/mL R-GalCer, 0.44 mg/mL F127), Tween20 micelles (5 mg/ mL Tween20), and R-GalCer-solubilized Tween20 micelles (0.22 mg/mL R-GalCer, 5 mg/mL Tween20). The R-GalCer/F127 dispersion was further diluted (15 times) by saline to eliminate the scattering (turbidity). Steady-state fluorescence anisotropy was measured with a Hitachi F-4500 spectrofluorometer (Tokyo) with an excitation wavelength of 360 nm and an emission wavelength of 430 nm through a Hoya L42 cutoff filter. The sample was put in a quartz cell and measured at 30 °C. The anisotropy value was determined by (IVV - GIVH)/(IVV + 2GIVH), where I indicates the fluorescence intensity, and the first and second subscripts denote the polarization direction (vertical or horizontal) of the excitation light and the analyzer, respectively. The grating correction factor G represents the ratio of the sensitivities of the detection system for vertically and horizontally polarized light and is given by IHV/IHH.
Results Particle Size and Stability of the Dispersions. The dispersions of R-GalCer were prepared by high-pressure emulsification using several nonionic surfactants as a
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Figure 2. Particle diameter of the dispersions as a function of the number of days after the preparation. The dispersions were prepared by high-pressure emulsification using Pluronic F68 (filled squares), F88 (open squares), F108 (filled circles), F127 (open circles), Tween 20 (filled diamonds), or Tween80 (open diamonds).
Figure 3. Small-angle (left) and wide-angle (right) X-ray diffraction patterns from the hydrated R-GalCer (a) and the dispersion of R-GalCer prepared with F127 (b).
stabilizer. The mean hydrodynamic diameter of the obtained particles is shown in Figure 2. Using Pluronic (F68, F88, F108, and F127), particles with the mean hydrodynamic diameter of 170-210 nm were obtained. The initial particle size depended somewhat on the molecular weight of Pluronic. The size did not change after 3 weeks, except for the case of F68, where a gradual increase in size was observed. For Pluronic L64 and P84, which are less hydrophilic than the others, the size of the prepared particles could not be determined because the dispersions immediately aggregated after preparation. Tween20 and 80 enabled the formation of particles with a similar initial size (200 nm); however, the stability of the dispersions was significantly lower than that of the dispersions prepared with Pluronic. The stable R-GalCer/ F127 dispersion was used to investigate the internal structure, as shown in the following sections. X-ray Diffraction. Figure 3 shows small-angle and wide-angle X-ray diffraction from the hydrated R-GalCer and R-GalCer/F127 dispersion. The small-angle diffraction from the hydrated R-GalCer represents a lamellar structure with a layer spacing of 61 Å. In the wide-angle diffraction, a peak at 1/4.2 Å-1 was observed, characteristic of the lateral packing of lipids in the gel phase. The diffraction from the R-GalCer/F127 dispersion was in accordance with that from the hydrated R-GalCer, suggesting a lamellar structure in the dispersed particles.
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Figure 4. DSC heating curves of the hydrated R-GalCer (a) and the dispersion of R-GalCer prepared with F127 (b).
Figure 5. Left: DSC cooling curves of the hydrated R-GalCer (a) and the hydrated R-GalCer/F127 mixture with the weight ratio of 1:20 (b). Right: DSC heating curves of the hydrated R-GalCer (a) and the hydrated R-GalCer/F127 mixtures with the weight ratios of 1:2 (b) and 1:20 (c). The curves are corrected for the R-GalCer mass.
DSC. The R-GalCer/F127 dispersion and the hydrated R-GalCer were analyzed by DSC as shown in Figure 4. The hydrated R-GalCer gave an endothermic peak with the apex at 74.9 °C representing the gel-liquid crystal (LC) phase transition. The R-GalCer/F127 dispersion also showed a peak at 73.4 °C. The peak was very broad, suggesting less cooperativity of the transition due to the fractionization (dispersion) of the phase. To investigate the effect of nonionic surfactants on the thermotropic behavior of the hydrated R-GalCer, the R-GalCer/F127 and R-GalCer/Tween20 hydrated mixtures were analyzed using DSC. As shown in Figure 5, the hydrated R-GalCer showed an exothermic or endothermic peak in a cooling or heating curve, respectively, corresponding to the gel-LC phase transition. In addition, mixing with F127 showed little change in both curves, suggesting R-GalCer and F127 are immiscible. On the other hand, Tween20 significantly changed the DSC curves. On cooling of the R-GalCer/Tween20 mixture, an exothermic peak appeared at lower temperature with a smaller peak area compared with the peak for R-GalCer (Figure 6, left). On heating, the R-GalCer/Tween20 mixture with the weight ratio of 1:2 gave an exothermic peak at 69.2 °C and a following endothermic peak at 75.0 °C (Figure 6, right (b)). A similar tendency was observed at the ratio of 1:20, but the position of both exothermic and endothermic peaks shifted to a lower temperature (Figure 6, right (c)). Reduction of the exothermic peak area on cooling and the appearance of the exothermic peak on heating suggest incomplete transition from the LC to
Formation of R-GalCer Dispersions
Figure 6. Left: DSC cooling curves of the hydrated R-GalCer (a) and the hydrated R-GalCer/Tween20 mixture with the weight ratio of 1:20 (b). Right: DSC heating curves of the hydrated R-GalCer (a) and the hydrated R-GalCer/Tween20 mixtures with the weight ratios of 1:2 (b), 1:20 (c), and 1:20 after annealing for 1 h at 50 °C (d). The curves are corrected for the R-GalCer mass.
gel phase. That is, a part of the LC phase transforms to the gel and the rest remains as a supercooled liquid crystal at low temperature, and the supercooled phase transforms to the gel on heating, followed by the gel-LC phase transition. Annealing the 1:20 mixture at 50 °C for 1 h caused the exothermic peak to disappear (Figure 6, right (d)), suggesting that the supercooled LC was completely transformed to the gel phase. The endothermic peak on heating became broader with an increase for Tween20, which suggests a reduction in the cooperativity of the transition due to the mixing of the gel phase with the surfactant. Solubility of r-GalCer in Micelles. The R-GalCer/ nonionic surfactant mixtures with the weight ratio of 2.2: 50 (approximately 1:20) in saline were repeatedly heated (to ca. 80 °C) and cooled (to room temperature) to check the solubility of R-GalCer in micelles. R-GalCer/Pluronic (F127, L64, P84, F68, F88, or F108) mixtures produced white precipitates at any temperature, suggesting that the Pluronic micelles could not solubilize R-GalCer. Even 10-fold higher amounts of F127 could not solubilize R-GalCer. On the other hand, the R-GalCer/Tween20 mixture dissolved and produced transparent solutions at ca. 80 °C, higher than the gel-LC transition temperature. The solution became slightly turbid as it was gradually cooled; however, it remained transparent when rapidly cooled to room temperature by running water. Although the R-GalCer/Tween80 solution became turbid at 80 °C because of the exceeding cloud point, rapid cooling provided a transparent solution at room temperature. These findings suggest that R-GalCer in the LC state, not in the gel state, is soluble in the Tween micelles and that R-GalCer in both the LC and gel states had little compatibility with Pluronic surfactants. Fluorescence Anisotropy. The steady-state fluorescence anisotropy of DPH in the R-GalCer/F127 dispersion was measured and compared with that in the micelles of Tween20 as shown in Figure 7. Evidently, the R-GalCer/ F127 dispersion had a higher anisotropy value than the Tween20 micelles with or without R-GalCer. Since a high anisotropy denotes that the fluorescence probe exists in a highly ordered environment, the present findings support the existence of the gel phases in the dispersions, revealed by small-angle X-ray scattering and DSC. Discussion Stable r-GalCer Dispersion Formation with Pluronic. The high-pressure emulsification produced the submicron particles. The type of surfactant somewhat
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Figure 7. Steady-state fluorescence anisotropy of DPH in several assemblies.
determines the particle size, but more importantly, it rather determines the stability of the dispersion. Pluronic L64 and P84, with longer hydrophobic (PPO) chains than hydrophilic (PEO) chains, could not produce stable dispersions. Other Pluronic and Tween surfactants are highly hydrophilic, but Pluronic had significantly higher ability to stabilize the dispersion than Tween. Surfactants with high hydrophilicity (high solubility into water) are expected to have high desorption rates from the interface, leading to destabilization of the kinetically stabilized dispersions. Nevertheless, Pluronic surfactants could hardly dissociate from the surface since they are of high molecular weight and their hydrophobic PPO chains can attach to the particle surface at more than one point, which intensively reduces the desorption rate. It has been shown that Pluronic F127 has a more pronounced effect than Tween80 on hindering the triglyceride degradation process of the solid lipid nanoparticles by pancreatic lipase.26 This result suggests that Pluronic has a more steric stabilization effect than Tween. The chemical structure of Pluronic is suggested to be effective in the stabilization of the R-GalCer dispersions. Higher r-GalCer Solubility in the Tween Micelles. In contrast to the dispersion stability discussed above, the solubility of R-GalCer was higher in the Tween micelles than in Pluronic micelles. This is attributed to the difference in the miscibility of R-GalCer with the surfactants. The DSC findings clearly show that R-GalCer and Pluronic are immiscible and separated in both gel and LC phases (Figure 5). Thus, it is reasonable that the Pluronic micelles cannot solubilize R-GalCer. For Tween, although its addition to R-GalCer makes the gel-LC transition less cooperative, they are not completely miscible since no significant decrease in the transition temperature was observed (Figure 6). In mixtures with dipalmitoylphosphatidylcholine, R-GalCer was miscible at the mole fraction of 0.3 and provided the transition temperature at ca. 45 °C and was immiscible at the fraction of 0.5 and transformed at ca. 70 °C.10 The solubility experiment with the Tween micelles revealed that the R-GalCer is soluble in the LC state but insoluble in the gel state. However, interestingly, the R-GalCer solubilized micellar solutions remained transparent when they were rapidly cooled from 80 °C. It is suggested from DSC findings that the rapid cooling produces the supercooled state of R-GalCer. The (26) Olbrich, C.; Mu¨ller, R. H. Int. J. Pharm. 1999, 180, 31-39.
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ability of the surfactants to provide the supercooled LC state has an important role for the solubilization of R-GalCer. Dispersion of the Gel Phase: A Novel Nanosuspension System. With Pluronic F127, a stable R-GalCer dispersion could be prepared. The dispersion provided a gel-LC transition temperature and diffraction patterns similar to those of the hydrated R-GalCer, suggesting the existence of the lamellar gel phase in the dispersed particles. The fluorescence anisotropy data also supported the existence of the gel phases in the dispersions. R-GalCer actually has two types of gel phases, the stable and the metastable gel, where the stable gel has a higher gel-LC transition temperature and more diffraction peaks than the metastable one.10 In the present study, the hydrated sample and the interior of the dispersed particles were metastable, considering their DSC and diffraction results.
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The findings of the present study demonstrated that the gel phase can be dispersed to form nanoparticles, in addition to the liquid, liquid crystal, and solid phases. In the nanoparticles, it is expected that R-GalCer can work either as a drug itself (nanosuspensions) or as a matrix in which other drugs can be contained (solid lipid nanoparticles). In cellular plasma membranes, glycosphingolipids are suggested to exist with cholesterol and other lipids with saturated fatty acids to form microdomains, where the glycolipids play important roles in intracellular and transmembrane signaling.27 Thus, this nanoparticle preparation can also be applied to these glycolipids with poor solubility, as a novel formula for controlling transmembrane signal transduction. LA0340605 (27) Mu¨ller, G. FEBS Lett. 2002, 531, 81-87.
