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Phase structure of liposome in lipid mixtures
Chemistry and Physics of Lipids 164 (2011) 722–726
Contents lists available at SciVerse ScienceDirect
Chemistry and Physics of Lipids journal homepage: www.elsevier.com/locate/chemphyslip
Phase structure of liposome in lipid mixtures Tianxi Zhang a,1 , Yuzhuo Li a , Anja Mueller b,∗ a b
Department of Chemistry, Clarkson University, Potsdam, NY 13699, USA Department of Chemistry, Central Michigan University, Mt. Pleasant, MI 48859, USA
a r t i c l e
i n f o
Article history: Received 8 April 2011 Received in revised form 11 August 2011 Accepted 12 August 2011 Available online 19 August 2011 Keywords: Lipids Liposome Aggregation Ultrasound image Phase structure
a b s t r a c t Gas microbubbles present in ultrasound imaging contrast agents are stabilized by lipid aggregates that typically contain a mixture of lipids. In this study, the phase structure of the lipid mixtures that contained two or three lipids was investigated using three different methods: dynamic light scattering, 1 H NMR, and microfluidity measurements with fluorescence probes. Three lipids that are commonly present in imaging agents (DPPC, DPPE-PEG, and DPPA) were used. Two types of systems, two-lipid model systems and simulated imaging systems were investigated. The results show that liposomes were the dominant aggregates in all the samples studied. The polar PEG side chains from the PEGylated lipid lead to the formation of micelles and micellar aggregates in small sizes. In the ternary lipid systems, almost all the lipids were present in bilayers with micelles absent and free lipids at very low concentration. These results suggest that liposomes, not micelles, contribute to the stabilization of microbubbles in an ultrasound imaging contrast agent. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Gas microbubbles are commonly present in an ultrasound imaging contrast agent for enhancement of backscattering of blood. The main cause of the enhancement in backscattering is the high compressibility of gas microbubbles relative to water (Phillips et al., 1998). In order to obtain a bright ultrasound image, many gas microbubbles should be present in a contrast agent. Dispersed microbubbles are readily eliminated in the capillary bed and crushed by the high pressure in the left ventricle. Thus, the stability of gas microbubbles is one of critical issues in the formulation of effective contrast agents. One of effective stabilizing agents for the gas microbubbles is to add various water-insoluble lipids (Phillips et al., 1998; De Jong and Ten Cate, 1996). Formation of lipid bilayer is preferred over micelles due to their much slower dynamic exchange rate among the lipid molecules involved. Size distribution and the number of over-sized of microbubbles have to be strictly controlled, as their size must be smaller than the diameter of the blood capillaries (4–8 m). The average particle size of approximately 1–2 m of the microbubbles is determined by the maximum response to the impinging ultrasound frequency. Similar to the situation in which
∗ Corresponding author. Tel.: +1 989 774 3956; fax: +1 989 774 3883. E-mail addresses: [email protected] (T. Zhang), [email protected] (A. Mueller). 1 Present address: Solix Biosystems, Inc., 430-B North College Avenue, Fort Collins, CO 80524, USA. Tel.: +1 970 692 5603; fax: +1 970 692 5669. 0009-3084/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2011.08.004
solid particles are used to stabilize an oil droplet in an oil-in-water emulsion, the most effective particle size for the stabilizing agent is about one tenth of the droplets to be stabilized. For the desired gas microbubble systems described above, the optimal particle size for a stabilizing agent would be in the range of 100–200 nm, which is in the range of liposomes (vesicles). For this imaging application, to avoid being filtered out by the reticuloendothelial system, liposome particles are generally stabilized by lipids (Woodle and Lasic, 1992). The gas phase is typically made from extremely hydrophobic perfluorocarbons (PFC), which have limited diffusivity in water. Thus greater stability of gas microbubbles (Frinking et al., 2000), interfacial interactions among the amphiphilic lipid bilayer, hydrophilic side chains in PEGylated lipid, and hydrophobic gas phase, are very complex. The mechanism of stabilization by interfacial interactions is not well documented. In order to have a clear physical picture of such complex interactions, it is essential to gain a fundamental understanding of the phase behavior of the lipid system in the presence of all those variables. In this study, the phase behavior of particular lipid mixtures commonly found in an ultrasound imaging contrast agent was investigated. More specifically, the focus is on the impact of two or three lipid mixtures and their alcoholic solvents on the related phase behavior. Three analytical techniques, including fluorescence, NMR, and dynamic light scattering, were used to characterize the phase structure. The knowledge generated from both simple model system and simulated imaging system provides insight into the stabilizing mechanism for a gas-containing system although perfluorocarbon is absent in the current study.
T. Zhang et al. / Chemistry and Physics of Lipids 164 (2011) 722–726
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2. Materials and methods
2.3. Instrumentation
2.1. Chemicals
Particle sizes were determined using dynamic light scattering (DLS) method on an ALV-NIBS/HPPS (Langen, Germany). A mode-selective fiber optical receiver was used for light processing. Single photons are converted by a sensitive single photon detector to electronic pulses. A real time Multiple Tau Digital Correlation Function (ALV-5000/EPP) is collected from these pulse-converted photos to get particle size information. The particle size (hydrodynamic radius) was calculated using the Stokes–Einstein equation (Johnsson and Edwards, 2003). Fluorescence spectra were recorded on a Perkin-Elmer fluorimeter (LS 50B) using ex of 320 nm and em of 350–450 nm. Slit widths were set as 3.5 nm for both excitation and emission. A stock solution of either pyrene (PY) or 1-pyreneacetic acid (PY-acetate) in ethanol was used. The ethanol was evaporated under nitrogen atmosphere and then under vacuum for 2 h. The final concentration of either PY or PY-acetate was 0.5 M in all samples. 1 H NMR spectra were performed on a Bruker 400 MHz spectrometer for all the samples.
Three different lipids that are typically used to stabilize gas microbubbles in an ultrasonic contrast agent were chosen in this study. They are 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphate sodium salt (DPPA), and 1,2-dipalmitoyl-sn-glycero-3phosphoethanolaine-N-[methoxy(polyethylene glycol)-5000] sodium salt (DPPE-PEG5000) and were purchased from Avanti Polar-Lipids. Pyrene (PY) and 1-pyreneacetic acid (PY-acetate) as fluorescence probes, propylene glycol, glycerol, and chloroform as organic solvents, deuterium oxide (D2 O containing 99.9% of D), and sodium chloride (analytical grade) were obtained from Sigma–Aldrich. All chemicals were used as received without further purification.
2.2. Lipid composition and sample preparation DPPC as one of three lipids studied herein has a glass transition temperature above body temperature, thus the solid-like structure of a DPPC bilayer decreases the gas exchange across the bilayer. The second lipid, negatively charged DPPA, prevents vesicle fusion to stabilize vesicle size. The third lipid, PEGylated DPPE-PEG5000, is needed to prevent uptake of the vesicles from the reticuloendothelial (RET) system of the body, aiming to increasing the lifetime of vesicles. Co-solvents are also needed in the formulation of the contrast agent in order to keep the hydrophobic perfluorocarbon gas in solution. Propylene glycol and glycerol are commonly used for this purpose. In order to elucidate the impact of lipid mixture and cosolvents on the lipid aggregation behavior, two types of mixture examples, the two-lipid model system and the simulated imaging systems, were studied in a total of five samples. Compositions of the five samples are presented in Table 1. Sample #1 and Sample #2 are model systems with two-lipids (DPPC and DPPE-PEG) in the absence of co-solvents to study the effect of lipid composition. Propylene glycol and glycerol are added in Sample #3 as co-solvents to mimic imaging systems. Sample #4 with three lipids (DPPC, DPPE-PEG, and DPPA) was to study the additional effect of charge stabilization. Sample #5 contains no lipids but the co-solvents as a control. All five samples contain 0.9 wt% of sodium chloride (NaCl) in D2 O which was used for NMR spectroscopy. Two-lipid model systems were prepared as Sample #1 and Sample #2. The lipids of DPPC and DPPE-PEG were dissolved in chloroform, and then the solvent was evaporated. The dried samples were suspended in NaCl (0.9 wt%) solution in D2 O. The total lipid concentrations were 10.0 mM in both samples. The final concentrations of DPPC and DPPE-PEG were 5.0 mM and 5.0 mM in Sample #1; 9.55 mM and 0.45 mM in Sample #2, respectively. The ratio of DPPC/DPPE-PEG varied from 1.0 in Sample #1 to 21.2 in Sample #2, respectively. This change affects the lipid aggregate structure (see more discussion in detail in the Section 3.1). The lipid suspensions were subjected to four heating-cooling cycles (65 ◦ C for 20 min and then room temperature) to obtain uniform samples. Preparation procedure of ultrasound imaging simulated systems as Sample #3 and Sample #4 was according to reference Phillips et al. (1998). Lipids were weighed and dissolved in propylene glycol (10%, v/v). The samples were heated at ∼69 ◦ C until the solutions were clear. Then glycerol (10%, w/v) and NaCl (0.9 wt%) in D2 O were added in the samples. The samples were heated at ∼69 ◦ C for 80 min, and then cooled to room temperature to get uniform samples.
3. Results and discussion 3.1. Two-lipid model systems Two different lipids are used in the simple model system containing a PEGylated-lipid. PEG side chains in DPPE-PEG lipid are necessary in a lipid-stabilized microbubble contrast agent as they give the particles stealth effect, which prevents the detection by the reticuloendothelial (RET) system (Woodle and Lasic, 1992). The introduction of the PEG group also significantly increases the effective volume of the polar head groups and hence affects the tendency of the lipids to form a bilayer structure (Koynova et al., 1999; Kenworthy et al., 1995). It is noted that pure PEGylated lipid in a solution forms micelles rather than bilayers. Similarly, addition of a PEGylated lipid into a membrane structure may eventually lead to phase separation: a segregation of bilayers from micelles. The exact point of segregation and the environmental impact on such a critical molar ratio for segregation is unknown. To elucidate such environmental and compositional effects, a model system that contains PEGylated-lipid and non-PEGylated lipid in varying ratios was investigated in this study. In Sample #1, the concentration of PEGylated lipid is high and thus it is expected that, in addition to liposomes, PEG-lipid micelles may also exist (Johnsson and Edwards, 2003; Johnsson et al., 2001). In Sample #2, with a much lower PEG-lipid-to-lipid ratio, liposomes are expected to be the dominating structure (Johnsson and Edwards, 2003). The difference in particle size distinguishes micelles from vesicles by DLS. The typical size of lipid based micelles is less than 50 nm. The size of commonly vesicles, on the other hand, is greater than 100 nm. In addition, it is expected that the lipid alkyl chains will not be observed in 1 H NMR spectrum when they are part of a bilayer as they move too slowly (Thurmond et al., 1996, 1997), while alkyl chains in free lipids and in micellar lipids should be visible. Note that 1 H NMR might not be sensitive enough to detect alkyl chains from free lipids at very low concentration, such as 10−10 M, in lipid mixtures that could form vesicles. Figs. 1 and 2 illustrate the particle size results of high and low PEG-lipid concentrations in Sample #1 and Sample #2, respectively. As expected, three peaks were present in Sample #1. They were assigned to micelles (8.5 nm), micellar aggregates (33.2 nm), and PEG-lipid liposomes (178 nm). In contrast, liposome-sized particles (166.2 nm) alone were seen in Sample #2. These particle size results indicated that PEG chains have a significant impact on the phase microstructure. Total concentrations of two lipids are the
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Table 1 Composition of lipid mixtures studied. Samplea
DPPC (mM)
DPPE-PEG (mM)
DPPA (mM)
Propylene glycol (%, v/v)
Glycerol (%, v/v)
#1 #2 #3 #4 #5
5.0 9.55 0.55 0.55 0
5.0 0.45 0.053 0.053 0
0 0 0 0.064 0
0 0 10.0 10.0 10.0
0 0 10.0 10.0 10.0
a
All samples contain 0.9 wt% of NaCl in D2 O.
Fig. 1. Particle sizes of two-lipid model system with high concentration of DPPEPEG in Sample #1. (The concentrations of DPPC and DPPE-PEG were 5.0 mM and 5.0 mM in Sample #1.)
same (10.0 mM) in both samples, only the ratio of DPPC/DPPE-PEG varied. The particle size results suggest that the high concentration of polar PEG-lipid leads to formation of micelles and micellar aggregates in small sizes. In order to further study the microstructure, 1 H NMR spectra of both two-lipid mixtures are summarized in Fig. 3. Both head group protons and PEG protons integrate according to their concentrations in each sample. The PEG resonance is a very narrow peak (3.78 ppm), suggesting that the PEG chain has free motion in all cases. In Sample #1, but not in Sample #2, broad resonances were also observed at 1.3 ppm and 0.89 ppm. These peaks were assigned as the hydrocarbon methylene groups [–(CH2 )n –] and terminal methyl groups (CH3 ) of the lipids, respectively. Since they are only visible in Sample #1 they are assigned to the lipids organized in micelles or micellar aggregates. Alkyl chains organized in a bilayer (possibly in the gel-phase) were not observed in Sample #2, likely due to restricted solvation or motion (Thurmond et al., 1996). The 1 H NMR results indeed confirm that polar PEG chains in the DPPE-PEG lipid tend to form micelles and micellar aggregates.
Fig. 3. 1 H NMR of two-lipid model systems of Sample #1 and Sample #2 in D2 O. (Inset shows enlargement of the alkyl region in Sample #1. Sample #2 does not show any peaks in that region.)
3.2. Simulated lipid imaging contrast agents Co-solvents and three lipids are commonly present in an imaging contrast agent. In this section, these two factors were investigated using Samples #3 and #4, mimicking lipid imaging contrast agents. Firstly, co-solvent properties could affect phase structure as a change of co-solvent polarity may influence lipid aggregation. Secondly, the third lipid, DPPA, is introduced in Sample #4. In addition to the steric repulsion from the PEG chains, DPPA provides charge repulsion. DPPA has a different glass transition temperature than DPPC and thus will also affect the phase behavior of the lipid mixture. The addition of small amounts of DPPA and PEG-lipid is a common method to stabilize liposomes and membranes towards fusion and aggregation (Hitzman et al., 2006; Woodle and Lasic, 1992). Figs. 4 and 5 illustrate the results of particle sizes from Sample #3 (with co-solvents, but without DPPA) and Sample #4 (with co-solvents and DPPA), respectively. Propylene glycol and glycerol were present in both samples as co-solvents. The particle size results are also summarized in Table 2. Sample #5 as a control
Table 2 Particle sizes of the simulated lipid systems.
Fig. 2. Particle size of two-lipid model system with low concentration of DPPEPEG in Sample #2. (The concentrations of DPPC and DPPE-PEG were 9.55 mM and 0.45 mM in Sample #2.)
Sample
Lipids
DPPA
Co-solvents
Size (nm)
#3 #4
2 3
No Yes
Yes Yes
22.1 (trace) 193.7 25.5 (trace) 109.5
T. Zhang et al. / Chemistry and Physics of Lipids 164 (2011) 722–726
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Table 3 Fluorescence peak ratios with pyrene from the simulated imaging systems.
Fig. 4. Particle sizes of the simulated lipid system without DPPA for Sample #3. (The concentrations of DPPC, DPPE-PEG, propylene glycol, and glycerol were 0.55 mM, 0.053 mM, 10% (v/v), and 10% (v/v) in Sample #3, respectively.)
(co-solvents alone without any lipids) did not show any particles, as expected for miscible solvents. In both samples, most of the particles were liposomes, with a small amount of micelles. Comparing Sample #2 to Sample #3, the same two lipids are used, but co-solvents were present in Sample #3. Co-solvents increased liposome size, from 193.7 nm in Sample #3 to 166.2 nm in Sample #2. Looking at micelles, the ratio of DPPC/DPPE-PEG in Sample #3 (10.4) was between those in Sample #1 (1.0) and Sample #2 (21.2). As discussed above, high DPPE-PEG lipid concentration tends to form micellar aggregates due to the polar PEG chains. Thus less micelles in Sample #3 were present than those in Sample #1, but more than those in Sample #2 (no observation of any micelles). When further comparing Sample #3 to Sample #4, DPPA is present in Sample #4, but not in Sample #3. The micelle size increases slightly from 22.1 nm to 25.5 nm with the addition of DPPA, and the liposome size decreases from 193.7 nm to 109.5 nm. The small amount of DPPA did not have a significant impact on the micellar aggregates as DPPA itself might not form micellar aggregates. It is also suggested that DPPA with charge repulsion tends to stabilize the liposome through size reduction. It is noted that total lipid concentrations are close to that in a real contrast agent in Sample #3 and Sample #4. To further confirm the structure designation, microfluidity of the particles was studied using pyrene and pyreneacetic acid (PYacetate) as fluorescence probes. Pyrene localizes in nonpolar center of a lipid bilayer (Heureux and Fragata, 1988), while amphiphilic PY-acetate is located at head-group–tail interface. Both are sensitive to changes in microfluidity of their environment (HAM effect) (Ramamurthy, 1991; Terzaghi et al., 1994; Antunes-Madeira and Madeira, 1993). Thus a change in the excimer (385 nm) to monomer
Sample ID
Lipids
DPPA
Co-solvents
III/I ratio
#3 #4 #5 D2 O
2 3 0 0
No Yes No No
Yes Yes Yes No
0.85 0.87 0.59 0.56
(372 nm) emission ratio (III/I ratio) can be used to determine a change from free lipid to micelles and liposomes. Table 3 summarizes the fluorescence results with pyrene from Samples #3, Sample #4, control Sample #5 (co-solvents alone), and D2 O as well. The low values of III/I ratio from co-solvent Sample #5 (0.59) and D2 O (0.56) suggest the absence of hydrophobic microenvironment, as there are no micelles or liposome aggregates in these two samples. The high III/I ratio for the lipid systems with co-solvents (0.85 in Sample #3 and 0.87 in Sample #4) indicates the presence of a hydrophobic microenvironment, suggesting bilayer aggregates. Similar values of the ratio between Sample #3 and Sample #4 also suggest that they have the same dominant microstructure. These fluorescence results confirm the designation of liposome structures in Samples #3 and #4. Fig. 6 shows the fluorescence results for Samples #1 through #4 and DPPE-PEG with dilute PY acetate. Sample #2 through Sample #4 was diluted beyond the critical micelle concentration (cmc) of DPPE-PEG (its cmc of about 30 M) (Priev et al., 2002), respectively, in order to prevent any interference from micelles. In all cases the ratios of III/I decreased with the dilution, indicating a decrease in the hydrophobic microenvironment. For Samples #3 and #4 the total lipid concentrations after 100-fold dilution were 6.0 M in Sample #3 and 6.7 M in Sample #4, respectively. The ratio values decreased close to the value of water (0.49) after the dilution, indicating that there was no hydrophobic microenvironment in the absence of either liposome or micelles in such dilute concentrations. The other three samples retained a more hydrophobic microenvironment after 100-fold dilution as their ratios of III/I were higher than 0.49. The DPPE-PEG sample after the dilution still had micellar structure as the lipid concentration of 60 M was higher than its cmc (30 M). The total lipid concentrations in Sample #1 and Sample #2 after the dilution were 100 M, which could be high enough to maintain liposome or micelles structures. Furthermore, 1 H NMR spectra were used to investigate the phase structure in the samples mimicking ultrasound imaging contrast agents. Solvents of d-propanediol and d-glycerol were compared as controls. Fig. 7 shows the 1 H NMR results from Samples #3 and #4 compared with both d-solvents alone. None of the samples showed any significant peaks at 0.89 ppm and 1.3 ppm assigned to
DPPE-PEG=0.6mM sample 1# sample 2# smaple 3# sample 4#
0.72
Peak ratio of III/I
0.68 0.64 0.60 0.56 0.52 0.48 0.44
Fig. 5. Particle sizes in the ternary lipid simulated system with DPPA for Sample #4. (The concentrations of DPPC, DPPE-PEG, DPPA, propylene glycol, and glycerol were 0.55 mM, 0.053 mM, 0.064 mM, 10% (v/v), and 10% (v/v) in Sample #4, respectively.)
0
20
40 60 Dilution times
80
100
Fig. 6. Fluorescence results with dilution using PY-acetate as probe.
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T. Zhang et al. / Chemistry and Physics of Lipids 164 (2011) 722–726
Acknowledgments The authors gratefully acknowledge the grant support in part for the research project from Bristol-Myers Squibb (BMS) Imaging, Inc. and insightful discussion with Dr. Robert Siegler of BMS. References
Fig. 7. 1 H NMR spectra of the simulated systems of Sample #3 and Sample #4 in D2 O, d-propanediol, and d-glycerol.
the hydrocarbon alkyl chains. This suggests that there were free lipids at very low concentration or trace micelles in the samples. These results further confirm that bilayer liposomes with the lipids were the dominating microstructure in Samples #3 and #4. 4. Conclusion For the two-lipid model systems, liposomes were the dominating microstructure. The polar PEG side chains from the DPPE-PEG had a significant impact on the phase microstructure. At high concentration of polar PEG-lipid, micelles and micellar aggregates are formed. For the simulated imaging systems, the presence of cosolvents did not have a significant influence on the phase structure. Almost all the lipids were found in bilayer liposomes, not micelles. These results suggest that the microbubbles in an ultrasound imaging agents are stabilized by lipid bilayers, not micelles. Further research on a real imaging contrast agent containing the hydrophobic gas will allow for a more detailed understanding of relationship between the phase structure of the stabilizing lipids and the stability of gas microbubble in an ultrasound imaging agents.
Antunes-Madeira, M., Madeira, V.M.C., 1993. Effects of DDE on the fluidity of model and native membranes: implications for the mechanisms of toxicity. Biochim. Biophys. Acta 1149, 86–92. De Jong, N., Ten Cate, F., 1996. New ultrasound contrast agents and technological innovations. Ultrasonics 34, 587–590. Frinking, P.J., Bouakaz, A., Kirkhorn, J., Ten Cate, F.J., 2000. Ultrasound contrast imaging: current and new potential methods. Ultrasound Med. Biol. 26, 965–975. Heureux, G.P.L., Fragata, M., 1988. Micropolarities of lipid bilayers and micelles 5. Localization of pyrene in small unilamellar phosphatidylcholine vesicles. Biophys. Chem. 30, 293–301. Hitzman, C.J., William, F.E., Wattenberg, L.W., Wiedmann, T.S., 2006. Development of a respirable, sustained release microcarrier for 5-fluorouracil I: in vitro assessment of liposomes, microspheres, and lipid coated nanoparticles. J. Pharm. Sci. 95, 1114–1126. Johnsson, M., Edwards, K., 2003. Liposomes, disks, and spherical micelles: aggregate structure in mixtures of gel phase phosphatidylcholines and poly(ethylene glycol)-phospholipids. Biophys. J. 85, 3839–3847. Johnsson, M., Hansson, P., Edwards, K., 2001. Spherical micelles and other selfassembled structures in dilute aqueous mixtures of poly(ethylene glycol) lipids. J. Phys. Chem. B 105, 8420–8430. Kenworthy, A.K., Simon, S.A., McIntosh, T.J., 1995. Structure and phase behavior of lipid suspensions containing phospholipids with covalently attached poly(ethylene glycol). Biophys. J. 68, 1903–1920. Koynova, R., Tenchov, B., Rapp, G., 1999. Effect of PEG-lipid conjugates on the phase behavior of phosphatidylethanolamine dispersions. Colloids Surf., A 149, 571–575. Phillips, D., Chen, X., Baggs, R., Rubens, D., Violaante, M., Parker, K., 1998. Acoustic backscatter properties of the particle/bubble ultrasound contrast agent. Ultrasonics 36, 883–892. Priev, A., Zamuel, S., Cohen, R., Barenholz, Y., 2002. Determination of critical micelle concentration of lipopolymers and other amphiphiles: comparison of sound velocity and fluorescent measurements. Langmuir 18, 612–617. Ramamurthy, V., 1991. Photochemistry in Organized and Constrained Media. VCH Publishers, New York, USA. Terzaghi, A., Tettamanti, G., Palestini, P., Acquotti, D., Bottiroli, G., Masserini, M., 1994. Fluorescence excimer formation imaging: a new technique to investigate association to cells and membrane behavior of glycolipids. Eur. J. Cell Biol. 65 (1), 172–177. Thurmond II, K.B., Kowalewski, T., Wooley, K.L., 1996. Water-soluble Knedel-like structures: the preparation of shell-cross-linked small particles. J. Am. Chem. Soc. 118, 7239–7240. Thurmond II, K.B, Kowalewski, T., Wooley, K.L., 1997. Shell cross-linked Knedels: a synthetic study of the factors affecting the dimensions and properties of amphiphilic core–shell nanospheres. J. Am. Chem. Soc. 119, 6656–6665. Woodle, M.C., Lasic, D.D., 1992. Sterically stabilized liposomes. Biochim. Biophys. Acta 1113, 171–199.
Contents lists available at SciVerse ScienceDirect
Chemistry and Physics of Lipids journal homepage: www.elsevier.com/locate/chemphyslip
Phase structure of liposome in lipid mixtures Tianxi Zhang a,1 , Yuzhuo Li a , Anja Mueller b,∗ a b
Department of Chemistry, Clarkson University, Potsdam, NY 13699, USA Department of Chemistry, Central Michigan University, Mt. Pleasant, MI 48859, USA
a r t i c l e
i n f o
Article history: Received 8 April 2011 Received in revised form 11 August 2011 Accepted 12 August 2011 Available online 19 August 2011 Keywords: Lipids Liposome Aggregation Ultrasound image Phase structure
a b s t r a c t Gas microbubbles present in ultrasound imaging contrast agents are stabilized by lipid aggregates that typically contain a mixture of lipids. In this study, the phase structure of the lipid mixtures that contained two or three lipids was investigated using three different methods: dynamic light scattering, 1 H NMR, and microfluidity measurements with fluorescence probes. Three lipids that are commonly present in imaging agents (DPPC, DPPE-PEG, and DPPA) were used. Two types of systems, two-lipid model systems and simulated imaging systems were investigated. The results show that liposomes were the dominant aggregates in all the samples studied. The polar PEG side chains from the PEGylated lipid lead to the formation of micelles and micellar aggregates in small sizes. In the ternary lipid systems, almost all the lipids were present in bilayers with micelles absent and free lipids at very low concentration. These results suggest that liposomes, not micelles, contribute to the stabilization of microbubbles in an ultrasound imaging contrast agent. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Gas microbubbles are commonly present in an ultrasound imaging contrast agent for enhancement of backscattering of blood. The main cause of the enhancement in backscattering is the high compressibility of gas microbubbles relative to water (Phillips et al., 1998). In order to obtain a bright ultrasound image, many gas microbubbles should be present in a contrast agent. Dispersed microbubbles are readily eliminated in the capillary bed and crushed by the high pressure in the left ventricle. Thus, the stability of gas microbubbles is one of critical issues in the formulation of effective contrast agents. One of effective stabilizing agents for the gas microbubbles is to add various water-insoluble lipids (Phillips et al., 1998; De Jong and Ten Cate, 1996). Formation of lipid bilayer is preferred over micelles due to their much slower dynamic exchange rate among the lipid molecules involved. Size distribution and the number of over-sized of microbubbles have to be strictly controlled, as their size must be smaller than the diameter of the blood capillaries (4–8 m). The average particle size of approximately 1–2 m of the microbubbles is determined by the maximum response to the impinging ultrasound frequency. Similar to the situation in which
∗ Corresponding author. Tel.: +1 989 774 3956; fax: +1 989 774 3883. E-mail addresses: [email protected] (T. Zhang), [email protected] (A. Mueller). 1 Present address: Solix Biosystems, Inc., 430-B North College Avenue, Fort Collins, CO 80524, USA. Tel.: +1 970 692 5603; fax: +1 970 692 5669. 0009-3084/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2011.08.004
solid particles are used to stabilize an oil droplet in an oil-in-water emulsion, the most effective particle size for the stabilizing agent is about one tenth of the droplets to be stabilized. For the desired gas microbubble systems described above, the optimal particle size for a stabilizing agent would be in the range of 100–200 nm, which is in the range of liposomes (vesicles). For this imaging application, to avoid being filtered out by the reticuloendothelial system, liposome particles are generally stabilized by lipids (Woodle and Lasic, 1992). The gas phase is typically made from extremely hydrophobic perfluorocarbons (PFC), which have limited diffusivity in water. Thus greater stability of gas microbubbles (Frinking et al., 2000), interfacial interactions among the amphiphilic lipid bilayer, hydrophilic side chains in PEGylated lipid, and hydrophobic gas phase, are very complex. The mechanism of stabilization by interfacial interactions is not well documented. In order to have a clear physical picture of such complex interactions, it is essential to gain a fundamental understanding of the phase behavior of the lipid system in the presence of all those variables. In this study, the phase behavior of particular lipid mixtures commonly found in an ultrasound imaging contrast agent was investigated. More specifically, the focus is on the impact of two or three lipid mixtures and their alcoholic solvents on the related phase behavior. Three analytical techniques, including fluorescence, NMR, and dynamic light scattering, were used to characterize the phase structure. The knowledge generated from both simple model system and simulated imaging system provides insight into the stabilizing mechanism for a gas-containing system although perfluorocarbon is absent in the current study.
T. Zhang et al. / Chemistry and Physics of Lipids 164 (2011) 722–726
723
2. Materials and methods
2.3. Instrumentation
2.1. Chemicals
Particle sizes were determined using dynamic light scattering (DLS) method on an ALV-NIBS/HPPS (Langen, Germany). A mode-selective fiber optical receiver was used for light processing. Single photons are converted by a sensitive single photon detector to electronic pulses. A real time Multiple Tau Digital Correlation Function (ALV-5000/EPP) is collected from these pulse-converted photos to get particle size information. The particle size (hydrodynamic radius) was calculated using the Stokes–Einstein equation (Johnsson and Edwards, 2003). Fluorescence spectra were recorded on a Perkin-Elmer fluorimeter (LS 50B) using ex of 320 nm and em of 350–450 nm. Slit widths were set as 3.5 nm for both excitation and emission. A stock solution of either pyrene (PY) or 1-pyreneacetic acid (PY-acetate) in ethanol was used. The ethanol was evaporated under nitrogen atmosphere and then under vacuum for 2 h. The final concentration of either PY or PY-acetate was 0.5 M in all samples. 1 H NMR spectra were performed on a Bruker 400 MHz spectrometer for all the samples.
Three different lipids that are typically used to stabilize gas microbubbles in an ultrasonic contrast agent were chosen in this study. They are 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphate sodium salt (DPPA), and 1,2-dipalmitoyl-sn-glycero-3phosphoethanolaine-N-[methoxy(polyethylene glycol)-5000] sodium salt (DPPE-PEG5000) and were purchased from Avanti Polar-Lipids. Pyrene (PY) and 1-pyreneacetic acid (PY-acetate) as fluorescence probes, propylene glycol, glycerol, and chloroform as organic solvents, deuterium oxide (D2 O containing 99.9% of D), and sodium chloride (analytical grade) were obtained from Sigma–Aldrich. All chemicals were used as received without further purification.
2.2. Lipid composition and sample preparation DPPC as one of three lipids studied herein has a glass transition temperature above body temperature, thus the solid-like structure of a DPPC bilayer decreases the gas exchange across the bilayer. The second lipid, negatively charged DPPA, prevents vesicle fusion to stabilize vesicle size. The third lipid, PEGylated DPPE-PEG5000, is needed to prevent uptake of the vesicles from the reticuloendothelial (RET) system of the body, aiming to increasing the lifetime of vesicles. Co-solvents are also needed in the formulation of the contrast agent in order to keep the hydrophobic perfluorocarbon gas in solution. Propylene glycol and glycerol are commonly used for this purpose. In order to elucidate the impact of lipid mixture and cosolvents on the lipid aggregation behavior, two types of mixture examples, the two-lipid model system and the simulated imaging systems, were studied in a total of five samples. Compositions of the five samples are presented in Table 1. Sample #1 and Sample #2 are model systems with two-lipids (DPPC and DPPE-PEG) in the absence of co-solvents to study the effect of lipid composition. Propylene glycol and glycerol are added in Sample #3 as co-solvents to mimic imaging systems. Sample #4 with three lipids (DPPC, DPPE-PEG, and DPPA) was to study the additional effect of charge stabilization. Sample #5 contains no lipids but the co-solvents as a control. All five samples contain 0.9 wt% of sodium chloride (NaCl) in D2 O which was used for NMR spectroscopy. Two-lipid model systems were prepared as Sample #1 and Sample #2. The lipids of DPPC and DPPE-PEG were dissolved in chloroform, and then the solvent was evaporated. The dried samples were suspended in NaCl (0.9 wt%) solution in D2 O. The total lipid concentrations were 10.0 mM in both samples. The final concentrations of DPPC and DPPE-PEG were 5.0 mM and 5.0 mM in Sample #1; 9.55 mM and 0.45 mM in Sample #2, respectively. The ratio of DPPC/DPPE-PEG varied from 1.0 in Sample #1 to 21.2 in Sample #2, respectively. This change affects the lipid aggregate structure (see more discussion in detail in the Section 3.1). The lipid suspensions were subjected to four heating-cooling cycles (65 ◦ C for 20 min and then room temperature) to obtain uniform samples. Preparation procedure of ultrasound imaging simulated systems as Sample #3 and Sample #4 was according to reference Phillips et al. (1998). Lipids were weighed and dissolved in propylene glycol (10%, v/v). The samples were heated at ∼69 ◦ C until the solutions were clear. Then glycerol (10%, w/v) and NaCl (0.9 wt%) in D2 O were added in the samples. The samples were heated at ∼69 ◦ C for 80 min, and then cooled to room temperature to get uniform samples.
3. Results and discussion 3.1. Two-lipid model systems Two different lipids are used in the simple model system containing a PEGylated-lipid. PEG side chains in DPPE-PEG lipid are necessary in a lipid-stabilized microbubble contrast agent as they give the particles stealth effect, which prevents the detection by the reticuloendothelial (RET) system (Woodle and Lasic, 1992). The introduction of the PEG group also significantly increases the effective volume of the polar head groups and hence affects the tendency of the lipids to form a bilayer structure (Koynova et al., 1999; Kenworthy et al., 1995). It is noted that pure PEGylated lipid in a solution forms micelles rather than bilayers. Similarly, addition of a PEGylated lipid into a membrane structure may eventually lead to phase separation: a segregation of bilayers from micelles. The exact point of segregation and the environmental impact on such a critical molar ratio for segregation is unknown. To elucidate such environmental and compositional effects, a model system that contains PEGylated-lipid and non-PEGylated lipid in varying ratios was investigated in this study. In Sample #1, the concentration of PEGylated lipid is high and thus it is expected that, in addition to liposomes, PEG-lipid micelles may also exist (Johnsson and Edwards, 2003; Johnsson et al., 2001). In Sample #2, with a much lower PEG-lipid-to-lipid ratio, liposomes are expected to be the dominating structure (Johnsson and Edwards, 2003). The difference in particle size distinguishes micelles from vesicles by DLS. The typical size of lipid based micelles is less than 50 nm. The size of commonly vesicles, on the other hand, is greater than 100 nm. In addition, it is expected that the lipid alkyl chains will not be observed in 1 H NMR spectrum when they are part of a bilayer as they move too slowly (Thurmond et al., 1996, 1997), while alkyl chains in free lipids and in micellar lipids should be visible. Note that 1 H NMR might not be sensitive enough to detect alkyl chains from free lipids at very low concentration, such as 10−10 M, in lipid mixtures that could form vesicles. Figs. 1 and 2 illustrate the particle size results of high and low PEG-lipid concentrations in Sample #1 and Sample #2, respectively. As expected, three peaks were present in Sample #1. They were assigned to micelles (8.5 nm), micellar aggregates (33.2 nm), and PEG-lipid liposomes (178 nm). In contrast, liposome-sized particles (166.2 nm) alone were seen in Sample #2. These particle size results indicated that PEG chains have a significant impact on the phase microstructure. Total concentrations of two lipids are the
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Table 1 Composition of lipid mixtures studied. Samplea
DPPC (mM)
DPPE-PEG (mM)
DPPA (mM)
Propylene glycol (%, v/v)
Glycerol (%, v/v)
#1 #2 #3 #4 #5
5.0 9.55 0.55 0.55 0
5.0 0.45 0.053 0.053 0
0 0 0 0.064 0
0 0 10.0 10.0 10.0
0 0 10.0 10.0 10.0
a
All samples contain 0.9 wt% of NaCl in D2 O.
Fig. 1. Particle sizes of two-lipid model system with high concentration of DPPEPEG in Sample #1. (The concentrations of DPPC and DPPE-PEG were 5.0 mM and 5.0 mM in Sample #1.)
same (10.0 mM) in both samples, only the ratio of DPPC/DPPE-PEG varied. The particle size results suggest that the high concentration of polar PEG-lipid leads to formation of micelles and micellar aggregates in small sizes. In order to further study the microstructure, 1 H NMR spectra of both two-lipid mixtures are summarized in Fig. 3. Both head group protons and PEG protons integrate according to their concentrations in each sample. The PEG resonance is a very narrow peak (3.78 ppm), suggesting that the PEG chain has free motion in all cases. In Sample #1, but not in Sample #2, broad resonances were also observed at 1.3 ppm and 0.89 ppm. These peaks were assigned as the hydrocarbon methylene groups [–(CH2 )n –] and terminal methyl groups (CH3 ) of the lipids, respectively. Since they are only visible in Sample #1 they are assigned to the lipids organized in micelles or micellar aggregates. Alkyl chains organized in a bilayer (possibly in the gel-phase) were not observed in Sample #2, likely due to restricted solvation or motion (Thurmond et al., 1996). The 1 H NMR results indeed confirm that polar PEG chains in the DPPE-PEG lipid tend to form micelles and micellar aggregates.
Fig. 3. 1 H NMR of two-lipid model systems of Sample #1 and Sample #2 in D2 O. (Inset shows enlargement of the alkyl region in Sample #1. Sample #2 does not show any peaks in that region.)
3.2. Simulated lipid imaging contrast agents Co-solvents and three lipids are commonly present in an imaging contrast agent. In this section, these two factors were investigated using Samples #3 and #4, mimicking lipid imaging contrast agents. Firstly, co-solvent properties could affect phase structure as a change of co-solvent polarity may influence lipid aggregation. Secondly, the third lipid, DPPA, is introduced in Sample #4. In addition to the steric repulsion from the PEG chains, DPPA provides charge repulsion. DPPA has a different glass transition temperature than DPPC and thus will also affect the phase behavior of the lipid mixture. The addition of small amounts of DPPA and PEG-lipid is a common method to stabilize liposomes and membranes towards fusion and aggregation (Hitzman et al., 2006; Woodle and Lasic, 1992). Figs. 4 and 5 illustrate the results of particle sizes from Sample #3 (with co-solvents, but without DPPA) and Sample #4 (with co-solvents and DPPA), respectively. Propylene glycol and glycerol were present in both samples as co-solvents. The particle size results are also summarized in Table 2. Sample #5 as a control
Table 2 Particle sizes of the simulated lipid systems.
Fig. 2. Particle size of two-lipid model system with low concentration of DPPEPEG in Sample #2. (The concentrations of DPPC and DPPE-PEG were 9.55 mM and 0.45 mM in Sample #2.)
Sample
Lipids
DPPA
Co-solvents
Size (nm)
#3 #4
2 3
No Yes
Yes Yes
22.1 (trace) 193.7 25.5 (trace) 109.5
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Table 3 Fluorescence peak ratios with pyrene from the simulated imaging systems.
Fig. 4. Particle sizes of the simulated lipid system without DPPA for Sample #3. (The concentrations of DPPC, DPPE-PEG, propylene glycol, and glycerol were 0.55 mM, 0.053 mM, 10% (v/v), and 10% (v/v) in Sample #3, respectively.)
(co-solvents alone without any lipids) did not show any particles, as expected for miscible solvents. In both samples, most of the particles were liposomes, with a small amount of micelles. Comparing Sample #2 to Sample #3, the same two lipids are used, but co-solvents were present in Sample #3. Co-solvents increased liposome size, from 193.7 nm in Sample #3 to 166.2 nm in Sample #2. Looking at micelles, the ratio of DPPC/DPPE-PEG in Sample #3 (10.4) was between those in Sample #1 (1.0) and Sample #2 (21.2). As discussed above, high DPPE-PEG lipid concentration tends to form micellar aggregates due to the polar PEG chains. Thus less micelles in Sample #3 were present than those in Sample #1, but more than those in Sample #2 (no observation of any micelles). When further comparing Sample #3 to Sample #4, DPPA is present in Sample #4, but not in Sample #3. The micelle size increases slightly from 22.1 nm to 25.5 nm with the addition of DPPA, and the liposome size decreases from 193.7 nm to 109.5 nm. The small amount of DPPA did not have a significant impact on the micellar aggregates as DPPA itself might not form micellar aggregates. It is also suggested that DPPA with charge repulsion tends to stabilize the liposome through size reduction. It is noted that total lipid concentrations are close to that in a real contrast agent in Sample #3 and Sample #4. To further confirm the structure designation, microfluidity of the particles was studied using pyrene and pyreneacetic acid (PYacetate) as fluorescence probes. Pyrene localizes in nonpolar center of a lipid bilayer (Heureux and Fragata, 1988), while amphiphilic PY-acetate is located at head-group–tail interface. Both are sensitive to changes in microfluidity of their environment (HAM effect) (Ramamurthy, 1991; Terzaghi et al., 1994; Antunes-Madeira and Madeira, 1993). Thus a change in the excimer (385 nm) to monomer
Sample ID
Lipids
DPPA
Co-solvents
III/I ratio
#3 #4 #5 D2 O
2 3 0 0
No Yes No No
Yes Yes Yes No
0.85 0.87 0.59 0.56
(372 nm) emission ratio (III/I ratio) can be used to determine a change from free lipid to micelles and liposomes. Table 3 summarizes the fluorescence results with pyrene from Samples #3, Sample #4, control Sample #5 (co-solvents alone), and D2 O as well. The low values of III/I ratio from co-solvent Sample #5 (0.59) and D2 O (0.56) suggest the absence of hydrophobic microenvironment, as there are no micelles or liposome aggregates in these two samples. The high III/I ratio for the lipid systems with co-solvents (0.85 in Sample #3 and 0.87 in Sample #4) indicates the presence of a hydrophobic microenvironment, suggesting bilayer aggregates. Similar values of the ratio between Sample #3 and Sample #4 also suggest that they have the same dominant microstructure. These fluorescence results confirm the designation of liposome structures in Samples #3 and #4. Fig. 6 shows the fluorescence results for Samples #1 through #4 and DPPE-PEG with dilute PY acetate. Sample #2 through Sample #4 was diluted beyond the critical micelle concentration (cmc) of DPPE-PEG (its cmc of about 30 M) (Priev et al., 2002), respectively, in order to prevent any interference from micelles. In all cases the ratios of III/I decreased with the dilution, indicating a decrease in the hydrophobic microenvironment. For Samples #3 and #4 the total lipid concentrations after 100-fold dilution were 6.0 M in Sample #3 and 6.7 M in Sample #4, respectively. The ratio values decreased close to the value of water (0.49) after the dilution, indicating that there was no hydrophobic microenvironment in the absence of either liposome or micelles in such dilute concentrations. The other three samples retained a more hydrophobic microenvironment after 100-fold dilution as their ratios of III/I were higher than 0.49. The DPPE-PEG sample after the dilution still had micellar structure as the lipid concentration of 60 M was higher than its cmc (30 M). The total lipid concentrations in Sample #1 and Sample #2 after the dilution were 100 M, which could be high enough to maintain liposome or micelles structures. Furthermore, 1 H NMR spectra were used to investigate the phase structure in the samples mimicking ultrasound imaging contrast agents. Solvents of d-propanediol and d-glycerol were compared as controls. Fig. 7 shows the 1 H NMR results from Samples #3 and #4 compared with both d-solvents alone. None of the samples showed any significant peaks at 0.89 ppm and 1.3 ppm assigned to
DPPE-PEG=0.6mM sample 1# sample 2# smaple 3# sample 4#
0.72
Peak ratio of III/I
0.68 0.64 0.60 0.56 0.52 0.48 0.44
Fig. 5. Particle sizes in the ternary lipid simulated system with DPPA for Sample #4. (The concentrations of DPPC, DPPE-PEG, DPPA, propylene glycol, and glycerol were 0.55 mM, 0.053 mM, 0.064 mM, 10% (v/v), and 10% (v/v) in Sample #4, respectively.)
0
20
40 60 Dilution times
80
100
Fig. 6. Fluorescence results with dilution using PY-acetate as probe.
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Acknowledgments The authors gratefully acknowledge the grant support in part for the research project from Bristol-Myers Squibb (BMS) Imaging, Inc. and insightful discussion with Dr. Robert Siegler of BMS. References
Fig. 7. 1 H NMR spectra of the simulated systems of Sample #3 and Sample #4 in D2 O, d-propanediol, and d-glycerol.
the hydrocarbon alkyl chains. This suggests that there were free lipids at very low concentration or trace micelles in the samples. These results further confirm that bilayer liposomes with the lipids were the dominating microstructure in Samples #3 and #4. 4. Conclusion For the two-lipid model systems, liposomes were the dominating microstructure. The polar PEG side chains from the DPPE-PEG had a significant impact on the phase microstructure. At high concentration of polar PEG-lipid, micelles and micellar aggregates are formed. For the simulated imaging systems, the presence of cosolvents did not have a significant influence on the phase structure. Almost all the lipids were found in bilayer liposomes, not micelles. These results suggest that the microbubbles in an ultrasound imaging agents are stabilized by lipid bilayers, not micelles. Further research on a real imaging contrast agent containing the hydrophobic gas will allow for a more detailed understanding of relationship between the phase structure of the stabilizing lipids and the stability of gas microbubble in an ultrasound imaging agents.
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