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Effects of a bolaamphiphile on the structure of phosphatidylcholine liposomes
Journal of Colloid and Interface Science 266 (2003) 442–447 www.elsevier.com/locate/jcis
Effects of a bolaamphiphile on the structure of phosphatidylcholine liposomes Qiang Gu,a Aihua Zou,a Chunwei Yuan,a,∗ and Rong Guo b a Key Laboratory of Molecular and Biomolecular Electronics (Southeast University), Ministry of Education, Nanjing 210096, China b Chemistry Department, Yangzhou University, Yangzhou 225002, China
Received 8 October 2002; accepted 9 June 2003
Abstract This paper describes the morphological characterization of aqueous dispersions of PC amphiphiles and bolaamphiphile AEC by microscopy, the liposomal membrane fluidity, and the zeta potential. Results indicate that the bolaamphiphile AEC can be included within conventional egg–PC liposome bilayers. This behavior could be due to their preference for the stretched conformation within the PC membranes. 2003 Elsevier Inc. All rights reserved. Keywords: Bolaamphiphile; PC liposomes; Ultraviolet spectrum; Zeta potential; Membrane fluidity; Micropolarity; Micrograph; MLM; Malachite green Abbreviations: PC, phosphatidylcholine; PBS, phosphate buffer saline; MLM, monolayer lipid membrane; MG, malachite green
1. Introduction Bolaamphiphilic lipids (bolaamphiphiles), which are known as cell-membrane components of archaebacteria [1,2], form highly stable monolayer membranes. The structures of archaebacterial membrane lipids are unusual in the same way as the environmental conditions in which these bacteria grow; extremely low pH values and high temperatures as well as high salt concentrations. Many studies have dealt with self-assembling morphologies of diverse bolaamphiphiles [3–5]. In particular, the bolaamphiphiles can form nonspherical molecular assemblies, such as nanoscale fibers [6,7], microtubules [8–11], and helical ribbons [12,13], with solid surfaces through intermolecular interactions between two hydrophilic headgroups [14]. The lipids of archacbacteria and also simpler model compounds are of great interest with regard to biotechnological research and material sciences. Moreover, one-chained bipolar compounds are also found in nature and show interesting biological activity. In recent years, much effort has been focused on in vivo drug carrier and delivery systems (liposomes, nanoparticles, etc.) that can escape removal from blood circulation * Corresponding author.
E-mail address: [email protected] (C.W. Yuan). 0021-9797/$ – see front matter 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0021-9797(03)00649-0
by the mononuclear phagocytic system and permit specific cell and organ targeting. Specific cell and organ targeting could be achieved by the elaboration of long-circulating liposomes that are further functionalized by ligands (sugars, immunoglobulins, proteins, etc.) specifically recognized by receptors present on the cell surface [15–17]. Despite these improvements, several obstacles limiting therapeutical applications of liposomes remain. The elaboration of liposomal devices with new or significantly improved properties requires the development of components that are substantially different from the phospholipids currently utilized. This paper is devoted to the polymorphic phase behavior in water of the amphiphile PC and bolaamphiphile displayed in Fig. 1. This study is a prerequisite when their use as liposomal components is intended. The liposomes they form with conventional phosphatidylcholine (PC) and bolaamphiphiles were analyzed by microscopy, liposomal membrane fluidity, and zeta potential. These are key issues when the use of liposomes as drug transport and delivery devices is contemplated. 2. Experimental 2.1. Materials Materials used included egg–PC (Microorganism Culture Medium Products Refinery of Haidian District, Beijing City,
Q. Gu et al. / Journal of Colloid and Interface Science 266 (2003) 442–447
Fig. 1. The structure of bolaamphiphile AEC.
China), pyrene (99%, Aldrich, America), and MG (malachite green) (Shanghai Chemical Reagent Company, China). Bolaamphiphile AEC (Fig. 1) was a kind gift from Professor Xu Jun (Research Institute of Jinling Petrochemical Corporation). Water used was distilled twice. 2.2. Preparation of liposomes A solution of PC in chloroform was placed in a 50-ml round-bottomed flask. Then the flask was transferred to a rotary evaporator and the solvent was removed under vacuum at 30 ◦ C. The resultant thin film of PC on the walls of the flask was dried under vacuum at room temperature for at least 2 h. The dry PC film was dispersed in phosphatebuffered saline (PBS). Then the system was sonicated under N2 for 30 min using a CQ250 water-bath-type sonicator (220 V, 50 Hz, from the Shanghai Ultrasonic Wave Instrument Works, Shanghai City, People’s Republic of China) to obtain liposomes. AEC bolaamphiphile was dissolved in phosphate buffer (pH 7.4), at a concentration of 1.02 × 10−3 mol/l. Then it was added to the liposome suspensions to prepare different weight ratios of AEC/PC samples, which were sonicated at 25 ◦ C. All the liposomes were observed using a JS94F microelectrophoretic mobility detector (Hua Dong Normal University, China). 2.3. UV measurement The UV spectrum was measured using a UV-2501 spectrophotometer (Shimadzu Corporation). 2.4. Determination of the micropolarity in PC liposomes The micropolarity in PC liposomes was determined by stable-state fluorometry with pyrene (1.3 × 10−6 mol/l) as probe on an RF-5301PC fluorescence spectrophotometer (Shimadzu Corporation). The intensity ratio of the first peak to the third peak (I1 /I3 ) of the fluorescence spectrum of pyrene shows micropolarity where the probe exists [17–19], which can show the structure changes of PC liposomes. The excited wavelength λex was 338 nm and the emission wavelength λem centered at 382 nm.
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fluidity upon AEC addition. The emission wavelength was fixed at 372 nm, and the sample was excited at 338 nm. The measurement of fluorescence polarization was carried out by inserting a polarization filter on the excitation and emission light paths of an RF-5301PC fluorescence spectrophotometer. The emission intensities of the polarized light, parallel and perpendicular to the excitation polarized light, I and I⊥ , were combined to give the following expression for steady-state polarization P [20]: P = [I − I⊥ ]/[I + I⊥ ].
(1)
2.6. Zeta potential measurement Zeta potential measurements were conducted using a JS94F microelectrophoretic mobility detector. The liposomes were injected into the rectangular quartz cell and then the instrument calculated the zeta potential automatically. Five zeta potential measurements were collected for each solution and the results averaged.
3. Results and discussions 3.1. The properties of the bolaamphiphile AEC The bolaamphiphile AEC (Fig. 1) was synthesized by the polymerization of 1,2-epoxypropane at high temperature and is an innocuous surfactant. In the bolaamphiphile, branched hydrocarbon chains and flexible groups of ether bonds characterize the hydrophobic regions. It has a low chaotic point like a nonionic surfactant, though it is an ionic surfactant. Moreover, this bipolar compound can form a monolayer lipid membrane (MLM, Fig. 2) when solubilized in water and shows interesting biological activity (unpublished observations). In contrast with vesicles formed by amphiphiles, MLM is formed by one layer of bolaamphiphiles and does not fuse. Because of its superior properties, we use it as an additive to PC liposomes and gain unusual phenomena.
2.5. Measurement of the liposomal membrane fluidity Pyrene solution in methanol was added to the liposomes suspensions to give a final concentration of 1.3 × 10−6 mol/l. Then the fluorescence polarization P of the probe was measured to characterize the change of the liposomal membrane
Fig. 2. Micrograph of MLM of 6 wt% AEC aqueous solution after sonication.
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Table 1 Values of the fluorescence micropolarity (I1 /I3 ) in PC liposomes with the addition of MG CMG (mol/l)
I1 /I3
0 1.0 × 10−5 2.0 × 10−5 3.0 × 10−5 4 × 10−5
1.033 1.040 1.063 1.067 1.073
Note. The concentration of PC liposomes is 3.2 × 10−4 mol/l.
3.2. The location of MG in PC liposomes 3.2.1. Micropolarity study of the PC liposomes system The location of MG in PC liposomes can be verified by the micropolarity of the PC liposomes determined by I1 /I3 of fluorescence spectra of pyrene. It is well known that pyrene exists at the interphase of liposomes where the polarity groups link hydrocarbon chain [18,19]. From Table 1, it can be seen that the value of I1 /I3 increases with the addition of MG. This result indicates that MG can be located in the palisade of PC liposomes because of the electrostatic interaction between the PC headgroup and MG molecule. The location of MG in the palisade of PC liposomes makes the spaces between the PC molecules in PC liposomes increase, which would lead to the transfer of pyrene to the outside of the liposome palisade and the increase of the microenvironment polarity of pyrene. In addition, the location of MG cations can increase the micropolarity around itself and so can the microenvironment polarity of pyrene. 3.2.2. The absorption of MG in PC liposomes According to the previous studies, MG cations can combine with hydroxyl ions and form alcohol in the alkaline solution, which has no absorption at 620 nm [21–23], so MG can fade in alkaline solution (Fig. 3). Hence, the absorption at 620 nm is the characteristic peak of the MG cation and the absorption peak at 260 nm is similar to that of the leuco structure, observed for MG in low-polarity solvents, such as 1,2-dimethoxyethane (DME) [24]. The absorption spectra of MG in PBS (pH of 7.4) aqueous solution and in the PC liposome system are shown in
Fig. 4. In PBS aqueous solution (Fig. 4, 1), the absorption bands of MG are observed at 260, 312, 425, and 620 nm. In PC liposomes (Fig. 4, 2), the absorbance at 260 nm increases, while the other absorption bands of MG decrease. These bands exhibit red shifts and an isosbestic point is observed at 280 nm. It is apparent that the number of MG existing in isomer b (Fig. 3) in the PC liposome system is more than that in PBS solution. The above results can be explained by the location of MG in PC liposomes. The trityl carbon cation center in the MG molecule is prone to interact with the PC headgroup, leading to the location of MG in the PC liposomes. So MG shows a fading effect in PC liposomes and the microenvironment around MG in PC liposomes should be much less polar than that in PBS aqueous solution [23]. Theoretically, the absorption wavelength is related to the droplet size. The larger the size of the droplets, the longer the absorption wavelength [25,26]. So the location of MG in PC liposomes results in the red shift of the absorption wavelength. From Fig. 4, it can be clearly seen that with the increase of the AEC content in mixed liposomes, the absorption peak
Fig. 4. The absorption spectra of MG (4.0 × 10−5 mol/l) at 25 ◦ C. Medium: (1) PBS aqueous solution (pH 7.4); (2) PC liposome system; (3–6) mixed liposomes systems. PC/AEC (mol/mol): (3) 6.4; (4) 3.2; (5) 2.1; (6) 1.6. PC concentration: 3.2 × 10−4 mol/l.
Fig. 3. The isomers of MG.
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Table 2 The change of fluorescence polarization (P ) and zeta potential of PC liposomes with the addition of AEC (25 ◦ C) Medium
P
ζ (mv)
PC PC/AEC = 6.4 (mol/mol) PC/AEC = 3.2 (mol/mol) PC/AEC = 2.1 (mol/mol) PC/AEC = 1.6 (mol/mol)
0.149 0.145 0.143 0.122 0.116
−16.1 −14.3 −10.2 −9.0 −7.6
Note. The concentration of PC is 3.2 × 10−4 mol/l.
formula [28], 1/P − 1/3 = (1/P0 − 1/3)(1 + KT τ/ηV ), Fig. 5. The fluorescence spectra of MG (4.0 × 10−5 mol/l) in PC/AEC liposomes at 25 ◦ C. PC/AEC (mol/mol): (2) pure PC; (3) 6.4; (4) 3.2; (5) 2.1; (6) 1.6. PC concentration: 3.2 × 10−4 mol/l.
of MG at 620 nm drops gradually while the peak intensity of 260 nm increases. Figure 5 shows that when excited at 260 nm, the intensity of the fluorescence emission peak of MG at 360 nm increases with the AEC content, which is consistent with the UV absorption. This is because the trityl carbon cation center in the MG molecule can also interact with the AEC anionic headgroup. And the location of MG in the palisade of the liposomes results in the decrease of the micropolarity and the increase of the environmental rigidity around MG molecules [27], which is also favorable to the existence of isomer b of MG. So it is obvious that with increased AEC content, mixed PC/AEC liposomes are formed and the incorporation of bolaamphiphile AEC in PC liposomes does not destroy the PC liposomes. PC is a zwitterionic lipid at the pH of the measurements (pH 7.4). Hence, as generally assumed there is not a strong electrostatic repulsion force between the amphoteric PC liposomes. Incorporation of bolaamphiphile AEC into the bilayer of PC liposomes increases the electrostatic repulsive force because AEC is a negatively charged lipid, whose presence in the liposomes enhanced the negative charge of the phosphate group. And thus the incorporation of bolaamphiphile AEC in PC liposomes enhances the stability of liposomes. 3.3. Fluorescence polarization study of the PC liposomes system In solutions of low viscosity, or in non-vitreous-state solutions, the rotation of molecular luminophores can change the orientation of the transition moment during the excited state lifetime. This induces an oscillator system, which can emit fluorescence deviating from the initial direction, and this produces fluorescence depolarization. The fluorescence depolarization is given by an expression known as the Perrin
(2)
where P0 is the limit polarization value when there is no molecular rotation or no interaction between the luminescent molecules and the adjacent ones (such as energy transfer or migration) in the system, K and T are the Boltzmann constant and the absolute temperature, respectively, τ is the fluorescent lifetime, V is the molecular volume, and η is the environmental viscosity of the luminescent molecules. Thus, if we know the values of P0 and τ and estimate the volume of the luminescent molecules, the viscosity of the media surrounding the probe can be calculated according to the P value measured. Fluorescent probes that can get into natural or artificial lipid bilayer membranes can be used to study their fluidity by the fluorescence polarization method [29]. In the present study, pyrene was also used as a fluorescent probe to study the fluidity of liposomal membranes, because it can easily get into the lipid bilayer. The fluorescent polarization of pyrene in PC liposomes was measured with different AEC content to characterize the change of liposomal membrane fluidity. The results are shown in Table 2. The value of P decreases with the addition of AEC. This result can be obtained only by the incorporation of bolaamphiphile AEC molecules into PC liposomes bilayers. Incorporating AEC into the bilayer causes the liposomes to change their packing geometrical structures. These geometrical changes include liposome size, the curvatures of the surface bilayer, and surface bilayer rigidity. It can also believe that the van der Waals forces, electrostatic repulsive forces, and hydration forces between the PC bilayers or PC liposomes will all be affected following the incorporation of AEC into the PC liposomes. So the doping of AEC bolaamphiphile on the PC liposomes changes the arrangement of PC molecules in the liposome microstructure and so decreases liposomal membrane fluidity (P ). Figure 6 shows the morphological character of PC liposomes by controlled bolaamphiphile AEC doping. From Fig. 6a, some PC liposomes can be easily seen. In contrast to Fig. 6a, the large vesicular objects (Figs. 6b and 6c) seem to consist of nets of connected small vesicles resulting from their partial fusion following sonication. This particular feature can be explained by the presence of unfracturable domains on the external layer of the liposomes. These unfracturable domains result very likely from segregation of
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(a) Fig. 7. The sketch map of the conformation of bolaamphiphile in PC liposomes. (a) U-comformation; (b) stretched conformation.
(b)
to the detriment of curved structures such as liposomes. This destabilizing effect could be due to their preference for the U-conformation (shown in Fig. 7) within the DMPC or DSPC membranes. So it can be suggested that the incorporation of bolaamphiphile AEC in the PC liposomes bilayer may adopt the stretched mode. And the reason that the bolaamphiphile AEC and PC can form mixed liposomes probably is the good miscibility of the binary AEC:PC mixture.
3.4. Zeta potential measurement
(c) Fig. 6. Micrograph of liposomes. (a) PC; (b) PC/AEC = 3.2 (mol/mol); (c) PC/AEC = 3.2 (mol/mol). PC concentration: 3.2 × 10−4 mol/l.
the lipids and could consist of bolaamphiphile molecules in a transmembrane conformation and/or of interdigitated PC molecules [30]. Formation of these domains suggests the fluid nature of vesicular microstructures, where lateral diffusion and reorganization readily occur. It is known that both general thermodynamic constraints and the geometry of each amphiphilic molecule present in a lipid matrix are crucial factors in determining the final shape and morphology of the aggregates formed [31]. Therefore, the morphological features of the PC/AEC mixed liposomes are determined by the property of these two lipids. In Figs. 6b and 6c, we also can see some liposomes like that in Fig. 6a, which seems to be in contradiction with the results obtained by Clary et al. [30]. Their results indicate that the fluorocarbon HO[C24 ][F6 C5 ]OH and hydrocarbon HO[C24 ][C12 ]OH bolaamphiphiles included within conventional DMPC or DSPC bilayers favor planar membranes
This study measured the zeta potential of the PC liposomes incorporated with various amounts of bolaamphiphile AEC. Table 2 reveals that the PC liposomes made of egg–PC possess negative charges at pH 7.4. This finding is consistent with that reported in literature [32,33], which indicates that there is a weak electrostatic repulsive force between the PC liposomes at pH 7.4. This experiment also demonstrates that the incorporation of bolaamphiphile into the PC bilayer decreases the negative zeta potential. The absolute value of the zeta potential of liposomes decreases from −16.1 to −7.6 mV as the concentration of AEC increases. In fact, PC is a zwitterionic lipid at the pH of the measurements (pH 7.4) and the zeta-potentials of PC liposomes are significantly smaller than those of the acidic phospholipid bilayer vesicles [32], while AEC is a negatively charged lipid, whose presence in the liposomes would enhance the negative charge of the phosphate group. So the AEC incorporation seems to increase the negative zeta potential and this contradicts with the results shown in Table 2. Thus, this phenomenon can only be explained by the exposed AEC chains shielding the negatively charged liposome surface, which decreases the absolute zeta potential of the mixed liposomes [34].
4. Conclusions Taken together, our results indicate that the bolaamphiphile AEC can be included within a conventional egg–PC
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liposome bilayer. This behavior could be due to its preference for the stretched conformation within the PC membranes, as suggested by the micrograph and other measurements. This result may be related to the miscibility of the binary AEC:PC mixture and the flexible group in AEC. As far as their applications as drug carriers and targeting devices are concerned, our results indicate that the bolaamphiphile AEC are most promising.
Acknowledgments This work is supported by the Project for Industrialization of Advanced and Novel Technology of University in Jiangsu Province (JH01-010) and the Natural Science Foundation of China (60121101).
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Effects of a bolaamphiphile on the structure of phosphatidylcholine liposomes Qiang Gu,a Aihua Zou,a Chunwei Yuan,a,∗ and Rong Guo b a Key Laboratory of Molecular and Biomolecular Electronics (Southeast University), Ministry of Education, Nanjing 210096, China b Chemistry Department, Yangzhou University, Yangzhou 225002, China
Received 8 October 2002; accepted 9 June 2003
Abstract This paper describes the morphological characterization of aqueous dispersions of PC amphiphiles and bolaamphiphile AEC by microscopy, the liposomal membrane fluidity, and the zeta potential. Results indicate that the bolaamphiphile AEC can be included within conventional egg–PC liposome bilayers. This behavior could be due to their preference for the stretched conformation within the PC membranes. 2003 Elsevier Inc. All rights reserved. Keywords: Bolaamphiphile; PC liposomes; Ultraviolet spectrum; Zeta potential; Membrane fluidity; Micropolarity; Micrograph; MLM; Malachite green Abbreviations: PC, phosphatidylcholine; PBS, phosphate buffer saline; MLM, monolayer lipid membrane; MG, malachite green
1. Introduction Bolaamphiphilic lipids (bolaamphiphiles), which are known as cell-membrane components of archaebacteria [1,2], form highly stable monolayer membranes. The structures of archaebacterial membrane lipids are unusual in the same way as the environmental conditions in which these bacteria grow; extremely low pH values and high temperatures as well as high salt concentrations. Many studies have dealt with self-assembling morphologies of diverse bolaamphiphiles [3–5]. In particular, the bolaamphiphiles can form nonspherical molecular assemblies, such as nanoscale fibers [6,7], microtubules [8–11], and helical ribbons [12,13], with solid surfaces through intermolecular interactions between two hydrophilic headgroups [14]. The lipids of archacbacteria and also simpler model compounds are of great interest with regard to biotechnological research and material sciences. Moreover, one-chained bipolar compounds are also found in nature and show interesting biological activity. In recent years, much effort has been focused on in vivo drug carrier and delivery systems (liposomes, nanoparticles, etc.) that can escape removal from blood circulation * Corresponding author.
E-mail address: [email protected] (C.W. Yuan). 0021-9797/$ – see front matter 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0021-9797(03)00649-0
by the mononuclear phagocytic system and permit specific cell and organ targeting. Specific cell and organ targeting could be achieved by the elaboration of long-circulating liposomes that are further functionalized by ligands (sugars, immunoglobulins, proteins, etc.) specifically recognized by receptors present on the cell surface [15–17]. Despite these improvements, several obstacles limiting therapeutical applications of liposomes remain. The elaboration of liposomal devices with new or significantly improved properties requires the development of components that are substantially different from the phospholipids currently utilized. This paper is devoted to the polymorphic phase behavior in water of the amphiphile PC and bolaamphiphile displayed in Fig. 1. This study is a prerequisite when their use as liposomal components is intended. The liposomes they form with conventional phosphatidylcholine (PC) and bolaamphiphiles were analyzed by microscopy, liposomal membrane fluidity, and zeta potential. These are key issues when the use of liposomes as drug transport and delivery devices is contemplated. 2. Experimental 2.1. Materials Materials used included egg–PC (Microorganism Culture Medium Products Refinery of Haidian District, Beijing City,
Q. Gu et al. / Journal of Colloid and Interface Science 266 (2003) 442–447
Fig. 1. The structure of bolaamphiphile AEC.
China), pyrene (99%, Aldrich, America), and MG (malachite green) (Shanghai Chemical Reagent Company, China). Bolaamphiphile AEC (Fig. 1) was a kind gift from Professor Xu Jun (Research Institute of Jinling Petrochemical Corporation). Water used was distilled twice. 2.2. Preparation of liposomes A solution of PC in chloroform was placed in a 50-ml round-bottomed flask. Then the flask was transferred to a rotary evaporator and the solvent was removed under vacuum at 30 ◦ C. The resultant thin film of PC on the walls of the flask was dried under vacuum at room temperature for at least 2 h. The dry PC film was dispersed in phosphatebuffered saline (PBS). Then the system was sonicated under N2 for 30 min using a CQ250 water-bath-type sonicator (220 V, 50 Hz, from the Shanghai Ultrasonic Wave Instrument Works, Shanghai City, People’s Republic of China) to obtain liposomes. AEC bolaamphiphile was dissolved in phosphate buffer (pH 7.4), at a concentration of 1.02 × 10−3 mol/l. Then it was added to the liposome suspensions to prepare different weight ratios of AEC/PC samples, which were sonicated at 25 ◦ C. All the liposomes were observed using a JS94F microelectrophoretic mobility detector (Hua Dong Normal University, China). 2.3. UV measurement The UV spectrum was measured using a UV-2501 spectrophotometer (Shimadzu Corporation). 2.4. Determination of the micropolarity in PC liposomes The micropolarity in PC liposomes was determined by stable-state fluorometry with pyrene (1.3 × 10−6 mol/l) as probe on an RF-5301PC fluorescence spectrophotometer (Shimadzu Corporation). The intensity ratio of the first peak to the third peak (I1 /I3 ) of the fluorescence spectrum of pyrene shows micropolarity where the probe exists [17–19], which can show the structure changes of PC liposomes. The excited wavelength λex was 338 nm and the emission wavelength λem centered at 382 nm.
443
fluidity upon AEC addition. The emission wavelength was fixed at 372 nm, and the sample was excited at 338 nm. The measurement of fluorescence polarization was carried out by inserting a polarization filter on the excitation and emission light paths of an RF-5301PC fluorescence spectrophotometer. The emission intensities of the polarized light, parallel and perpendicular to the excitation polarized light, I and I⊥ , were combined to give the following expression for steady-state polarization P [20]: P = [I − I⊥ ]/[I + I⊥ ].
(1)
2.6. Zeta potential measurement Zeta potential measurements were conducted using a JS94F microelectrophoretic mobility detector. The liposomes were injected into the rectangular quartz cell and then the instrument calculated the zeta potential automatically. Five zeta potential measurements were collected for each solution and the results averaged.
3. Results and discussions 3.1. The properties of the bolaamphiphile AEC The bolaamphiphile AEC (Fig. 1) was synthesized by the polymerization of 1,2-epoxypropane at high temperature and is an innocuous surfactant. In the bolaamphiphile, branched hydrocarbon chains and flexible groups of ether bonds characterize the hydrophobic regions. It has a low chaotic point like a nonionic surfactant, though it is an ionic surfactant. Moreover, this bipolar compound can form a monolayer lipid membrane (MLM, Fig. 2) when solubilized in water and shows interesting biological activity (unpublished observations). In contrast with vesicles formed by amphiphiles, MLM is formed by one layer of bolaamphiphiles and does not fuse. Because of its superior properties, we use it as an additive to PC liposomes and gain unusual phenomena.
2.5. Measurement of the liposomal membrane fluidity Pyrene solution in methanol was added to the liposomes suspensions to give a final concentration of 1.3 × 10−6 mol/l. Then the fluorescence polarization P of the probe was measured to characterize the change of the liposomal membrane
Fig. 2. Micrograph of MLM of 6 wt% AEC aqueous solution after sonication.
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Table 1 Values of the fluorescence micropolarity (I1 /I3 ) in PC liposomes with the addition of MG CMG (mol/l)
I1 /I3
0 1.0 × 10−5 2.0 × 10−5 3.0 × 10−5 4 × 10−5
1.033 1.040 1.063 1.067 1.073
Note. The concentration of PC liposomes is 3.2 × 10−4 mol/l.
3.2. The location of MG in PC liposomes 3.2.1. Micropolarity study of the PC liposomes system The location of MG in PC liposomes can be verified by the micropolarity of the PC liposomes determined by I1 /I3 of fluorescence spectra of pyrene. It is well known that pyrene exists at the interphase of liposomes where the polarity groups link hydrocarbon chain [18,19]. From Table 1, it can be seen that the value of I1 /I3 increases with the addition of MG. This result indicates that MG can be located in the palisade of PC liposomes because of the electrostatic interaction between the PC headgroup and MG molecule. The location of MG in the palisade of PC liposomes makes the spaces between the PC molecules in PC liposomes increase, which would lead to the transfer of pyrene to the outside of the liposome palisade and the increase of the microenvironment polarity of pyrene. In addition, the location of MG cations can increase the micropolarity around itself and so can the microenvironment polarity of pyrene. 3.2.2. The absorption of MG in PC liposomes According to the previous studies, MG cations can combine with hydroxyl ions and form alcohol in the alkaline solution, which has no absorption at 620 nm [21–23], so MG can fade in alkaline solution (Fig. 3). Hence, the absorption at 620 nm is the characteristic peak of the MG cation and the absorption peak at 260 nm is similar to that of the leuco structure, observed for MG in low-polarity solvents, such as 1,2-dimethoxyethane (DME) [24]. The absorption spectra of MG in PBS (pH of 7.4) aqueous solution and in the PC liposome system are shown in
Fig. 4. In PBS aqueous solution (Fig. 4, 1), the absorption bands of MG are observed at 260, 312, 425, and 620 nm. In PC liposomes (Fig. 4, 2), the absorbance at 260 nm increases, while the other absorption bands of MG decrease. These bands exhibit red shifts and an isosbestic point is observed at 280 nm. It is apparent that the number of MG existing in isomer b (Fig. 3) in the PC liposome system is more than that in PBS solution. The above results can be explained by the location of MG in PC liposomes. The trityl carbon cation center in the MG molecule is prone to interact with the PC headgroup, leading to the location of MG in the PC liposomes. So MG shows a fading effect in PC liposomes and the microenvironment around MG in PC liposomes should be much less polar than that in PBS aqueous solution [23]. Theoretically, the absorption wavelength is related to the droplet size. The larger the size of the droplets, the longer the absorption wavelength [25,26]. So the location of MG in PC liposomes results in the red shift of the absorption wavelength. From Fig. 4, it can be clearly seen that with the increase of the AEC content in mixed liposomes, the absorption peak
Fig. 4. The absorption spectra of MG (4.0 × 10−5 mol/l) at 25 ◦ C. Medium: (1) PBS aqueous solution (pH 7.4); (2) PC liposome system; (3–6) mixed liposomes systems. PC/AEC (mol/mol): (3) 6.4; (4) 3.2; (5) 2.1; (6) 1.6. PC concentration: 3.2 × 10−4 mol/l.
Fig. 3. The isomers of MG.
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Table 2 The change of fluorescence polarization (P ) and zeta potential of PC liposomes with the addition of AEC (25 ◦ C) Medium
P
ζ (mv)
PC PC/AEC = 6.4 (mol/mol) PC/AEC = 3.2 (mol/mol) PC/AEC = 2.1 (mol/mol) PC/AEC = 1.6 (mol/mol)
0.149 0.145 0.143 0.122 0.116
−16.1 −14.3 −10.2 −9.0 −7.6
Note. The concentration of PC is 3.2 × 10−4 mol/l.
formula [28], 1/P − 1/3 = (1/P0 − 1/3)(1 + KT τ/ηV ), Fig. 5. The fluorescence spectra of MG (4.0 × 10−5 mol/l) in PC/AEC liposomes at 25 ◦ C. PC/AEC (mol/mol): (2) pure PC; (3) 6.4; (4) 3.2; (5) 2.1; (6) 1.6. PC concentration: 3.2 × 10−4 mol/l.
of MG at 620 nm drops gradually while the peak intensity of 260 nm increases. Figure 5 shows that when excited at 260 nm, the intensity of the fluorescence emission peak of MG at 360 nm increases with the AEC content, which is consistent with the UV absorption. This is because the trityl carbon cation center in the MG molecule can also interact with the AEC anionic headgroup. And the location of MG in the palisade of the liposomes results in the decrease of the micropolarity and the increase of the environmental rigidity around MG molecules [27], which is also favorable to the existence of isomer b of MG. So it is obvious that with increased AEC content, mixed PC/AEC liposomes are formed and the incorporation of bolaamphiphile AEC in PC liposomes does not destroy the PC liposomes. PC is a zwitterionic lipid at the pH of the measurements (pH 7.4). Hence, as generally assumed there is not a strong electrostatic repulsion force between the amphoteric PC liposomes. Incorporation of bolaamphiphile AEC into the bilayer of PC liposomes increases the electrostatic repulsive force because AEC is a negatively charged lipid, whose presence in the liposomes enhanced the negative charge of the phosphate group. And thus the incorporation of bolaamphiphile AEC in PC liposomes enhances the stability of liposomes. 3.3. Fluorescence polarization study of the PC liposomes system In solutions of low viscosity, or in non-vitreous-state solutions, the rotation of molecular luminophores can change the orientation of the transition moment during the excited state lifetime. This induces an oscillator system, which can emit fluorescence deviating from the initial direction, and this produces fluorescence depolarization. The fluorescence depolarization is given by an expression known as the Perrin
(2)
where P0 is the limit polarization value when there is no molecular rotation or no interaction between the luminescent molecules and the adjacent ones (such as energy transfer or migration) in the system, K and T are the Boltzmann constant and the absolute temperature, respectively, τ is the fluorescent lifetime, V is the molecular volume, and η is the environmental viscosity of the luminescent molecules. Thus, if we know the values of P0 and τ and estimate the volume of the luminescent molecules, the viscosity of the media surrounding the probe can be calculated according to the P value measured. Fluorescent probes that can get into natural or artificial lipid bilayer membranes can be used to study their fluidity by the fluorescence polarization method [29]. In the present study, pyrene was also used as a fluorescent probe to study the fluidity of liposomal membranes, because it can easily get into the lipid bilayer. The fluorescent polarization of pyrene in PC liposomes was measured with different AEC content to characterize the change of liposomal membrane fluidity. The results are shown in Table 2. The value of P decreases with the addition of AEC. This result can be obtained only by the incorporation of bolaamphiphile AEC molecules into PC liposomes bilayers. Incorporating AEC into the bilayer causes the liposomes to change their packing geometrical structures. These geometrical changes include liposome size, the curvatures of the surface bilayer, and surface bilayer rigidity. It can also believe that the van der Waals forces, electrostatic repulsive forces, and hydration forces between the PC bilayers or PC liposomes will all be affected following the incorporation of AEC into the PC liposomes. So the doping of AEC bolaamphiphile on the PC liposomes changes the arrangement of PC molecules in the liposome microstructure and so decreases liposomal membrane fluidity (P ). Figure 6 shows the morphological character of PC liposomes by controlled bolaamphiphile AEC doping. From Fig. 6a, some PC liposomes can be easily seen. In contrast to Fig. 6a, the large vesicular objects (Figs. 6b and 6c) seem to consist of nets of connected small vesicles resulting from their partial fusion following sonication. This particular feature can be explained by the presence of unfracturable domains on the external layer of the liposomes. These unfracturable domains result very likely from segregation of
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(a) Fig. 7. The sketch map of the conformation of bolaamphiphile in PC liposomes. (a) U-comformation; (b) stretched conformation.
(b)
to the detriment of curved structures such as liposomes. This destabilizing effect could be due to their preference for the U-conformation (shown in Fig. 7) within the DMPC or DSPC membranes. So it can be suggested that the incorporation of bolaamphiphile AEC in the PC liposomes bilayer may adopt the stretched mode. And the reason that the bolaamphiphile AEC and PC can form mixed liposomes probably is the good miscibility of the binary AEC:PC mixture.
3.4. Zeta potential measurement
(c) Fig. 6. Micrograph of liposomes. (a) PC; (b) PC/AEC = 3.2 (mol/mol); (c) PC/AEC = 3.2 (mol/mol). PC concentration: 3.2 × 10−4 mol/l.
the lipids and could consist of bolaamphiphile molecules in a transmembrane conformation and/or of interdigitated PC molecules [30]. Formation of these domains suggests the fluid nature of vesicular microstructures, where lateral diffusion and reorganization readily occur. It is known that both general thermodynamic constraints and the geometry of each amphiphilic molecule present in a lipid matrix are crucial factors in determining the final shape and morphology of the aggregates formed [31]. Therefore, the morphological features of the PC/AEC mixed liposomes are determined by the property of these two lipids. In Figs. 6b and 6c, we also can see some liposomes like that in Fig. 6a, which seems to be in contradiction with the results obtained by Clary et al. [30]. Their results indicate that the fluorocarbon HO[C24 ][F6 C5 ]OH and hydrocarbon HO[C24 ][C12 ]OH bolaamphiphiles included within conventional DMPC or DSPC bilayers favor planar membranes
This study measured the zeta potential of the PC liposomes incorporated with various amounts of bolaamphiphile AEC. Table 2 reveals that the PC liposomes made of egg–PC possess negative charges at pH 7.4. This finding is consistent with that reported in literature [32,33], which indicates that there is a weak electrostatic repulsive force between the PC liposomes at pH 7.4. This experiment also demonstrates that the incorporation of bolaamphiphile into the PC bilayer decreases the negative zeta potential. The absolute value of the zeta potential of liposomes decreases from −16.1 to −7.6 mV as the concentration of AEC increases. In fact, PC is a zwitterionic lipid at the pH of the measurements (pH 7.4) and the zeta-potentials of PC liposomes are significantly smaller than those of the acidic phospholipid bilayer vesicles [32], while AEC is a negatively charged lipid, whose presence in the liposomes would enhance the negative charge of the phosphate group. So the AEC incorporation seems to increase the negative zeta potential and this contradicts with the results shown in Table 2. Thus, this phenomenon can only be explained by the exposed AEC chains shielding the negatively charged liposome surface, which decreases the absolute zeta potential of the mixed liposomes [34].
4. Conclusions Taken together, our results indicate that the bolaamphiphile AEC can be included within a conventional egg–PC
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liposome bilayer. This behavior could be due to its preference for the stretched conformation within the PC membranes, as suggested by the micrograph and other measurements. This result may be related to the miscibility of the binary AEC:PC mixture and the flexible group in AEC. As far as their applications as drug carriers and targeting devices are concerned, our results indicate that the bolaamphiphile AEC are most promising.
Acknowledgments This work is supported by the Project for Industrialization of Advanced and Novel Technology of University in Jiangsu Province (JH01-010) and the Natural Science Foundation of China (60121101).
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