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Polymer-stabilized phospholipid vesicles formed on polyelectrolyte multilayer capsules
BBRC Biochemical and Biophysical Research Communications 303 (2003) 653–659 www.elsevier.com/locate/ybbrc
Polymer-stabilized phospholipid vesicles formed on polyelectrolyte multilayer capsules Liqin Ge,a Helmuth M€ ohwald,b and Junbai Lia,* a
International Joint Lab, The Center for Molecular Science, Institute of Chemistry, Chinese Academy of Science, Beijing 100080, China b Max-Planck-Institute of Colloids and Interfaces, Am Muehlenberg 2, Golm/Potsdam D-14476, Germany Received 18 February 2003
Abstract Phospholipid vesicles on polyelectrolyte multilayer shells can be stabilized against ethanol by coating a single cationic polyelectrolyte. Confocal laser scanning microscopy (CLSM) proved that the lipids were stabilized by cationic polyelectrolytes and the permeability to small hydrophilic dyes was decreased. Measurements of fluorescence recovery after photo-bleaching (FRAP) with individual capsules enable quantification of release profiles. Ó 2003 Published by Elsevier Science (USA). Keywords: Release; Vesicles; Phospholipids; Polyelectrolyte shells; Polymer complex; CLSM
Phospholipid vesicles have received much attention as possible drug carriers and model systems to study biological processes at membranes [1–5]. In many practical applications, however, they lack the stable natural system, which is gained by coating of biopolymers on both sides of the membrane, and which is difficult to realize [3]. Consequently, these attempts to stabilize the bilayer via polymerization were mostly unsuccessful due to lateral phase separation [4–6]. As an alternative, one may prepare vesicles by adsorbing polyelectrolytes on both sides and a variant of this approach has been tested [7,8]. In order to prepare stabilized vesicles with a defined size, an alternative strategy has recently been developed by employing layer-by-layer technique. Polyelectrolyte multilayers were prepared on colloidal particles that were later sacrificed by dissolution to obtain hollow capsules. On these capsules a phospholipid bilayer is adsorbed, thereafter, a polycation layer was coated. In this way, one can obtain vesicles with a defined size, shape, and achieve some stability [9–17]. An understanding of such complex vesicle characteristics is not only of scientific interest but is also motivated by release applications [15,18]. It has been shown * Corresponding author. Fax: +86-10-8261-2484. E-mail address: [email protected] (J. Li).
from our previous studies that polyelectrolytes can stabilize liposomes [19]. Five layer ðPSS=PAHÞ5 capsules are one of the most frequently investigated systems. Thus we try to make use of polyelectrolytes to modify the lipid/capsule complex vesicles. The fabrication procedure and the stabilization mechanisms of the ðPSS=PAHÞ5 =DMPA=PAH vesicles are demonstrated in the following Scheme 1A and B, respectively.
Materials and methods Materials. The sources of chemicals were as follows: poly(styrenesulfonate, sodium salt) (PSS, Mw 70,000) and poly(allylamine hydrochloride) (PAH, Mw 70,000) were from Aldrich. L -a-Dimyristoylphosphatidic acid (DMPA), 6-carboxyfluorescein (6-CF), fluorescently labeled DPPC (L -a-phosphatidylcholine (NBD-aminohexanoyl)-c-palmitoyl) (NBD-DPPC), and ethanol were purchased from Sigma. Two ethanol solutions were prepared with different ratios: VEtOH :Vwater ¼ 1:10 and 1:1. All these materials were used as received. PAH was covalently labeled by rhodamine isothiocyanate (molecular probes) based on [20]. The molecular structures of the compounds used in this study are shown in Scheme 2. The water used in all experiments was prepared in a three-stage Millipore Milli-Q Plus 185 purification system and had a resistivity higher than 18:2 MX cm1 . Positively charged melamine formaldehyde particles (MF particles) with a diameter of around 2:85 0:09 lm were obtained from Microparticles GmbH, Berlin.
0006-291X/03/$ - see front matter Ó 2003 Published by Elsevier Science (USA). doi:10.1016/S0006-291X(03)00391-7
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Scheme 1. Illustration of procedures for preparing the ðPSS=PAHÞ5 =DMPA vesicles with a further coating by polyelectrolytes (A) and the stabilization of test of the complex vesicles (B).
Scheme 2. Molecular structures of the compounds used in this study. Polyelectrolyte shells prepared through layer-by-layer adsorption. Multilayer assembly was accomplished by the adsorption of polyelectrolytes at a concentration of 1 mg/ml, 0.5 M NaCl aqueous solutions. Oppositely charged polyelectrolyte species were subsequently added to the suspension followed by repeated centrifugation cycles. After the intended number of layers was adsorbed, 0.1 M HCl was used to remove the core (MF particles) and the obtained hollow polyelectrolyte shells were obtained [14]. PSS is used to form the first layer and the outermost layer is PAH, which has a positive charge in order to bind the negatively charged phospholipids in the next step. Preparation of phospholipid solution. The phospholipid, DMPA, was dissolved in a mixed solvent of chloroform and methanol (VCHCl3 :VMeOH ¼ 1:1) with a concentration of 0.5 mg/ml. Then the solvent was evaporated in a rotavap at 30 °C, and afterwards water was added to a final lipid concentration (CDMPA ¼ 0:025 mg=ml), and sonicated for 5 min. Lipid solution was added into polyelectrolyte shells and allowed 5 min for adsorption. Then the mixed solution was washed for at least three times with water to remove the non-adsorbed materials by centrifugation.
Measurements of CLSM. The images of capsules were obtained by a Leica confocal scanning system. A 100 oil immersion objective with a numerical aperture of 1.4 was used. In the experiment, we used fluorescently labeled NBD-DPPC at 5 wt%. 6-Carboxyfluorescein (6-CF) and rhodamine b-PAH (Mw 65,000) were selected as hydrophilic fluorescence dyes. Measurements of FRAP. In order to study the penetration of dyes across the wall, the dyes inside the capsules were photo-chemically bleached. To do this, an argon ion laser from the CLSM emitting at a wavelength of k ¼ 488 nm was used. The laser beam was focused onto selected area inside the capsule. The time for bleaching was long enough to ensure that almost all dye molecules in the capsule were bleached. Imaging was typically performed at a rather low laser intensity. The interval between each image scan varied with the duration of recovery established at an initial pilot experiment. Recovery was considered complete when the intensity of the photo-bleached region was stable and the curve was flat. For quantitative analysis, the fluorescence intensity was integrated by tracing a fixed area in the interior (ROI analysis system provided by the CLSM software), giving an intensity value for each time point. We have to point out here that there is always a small amount of fluorescence dyes lost during repeatedly scanning and recovering. However, this will not influence the experimental results. Electrophoretic mobility measurements. The zeta-potential of the capsules was measured by a Malvern Zetasizer 4. The mobility l was converted into a zeta (f)-potential by the Smoluchowski relation, f ¼ lg=e, where g and e are the viscosity and permeability of the solution, respectively [14].
Results and discussion Assembly of phospholipids vesicles onto polyelectrolyte capsules To compare quantitatively the fluorescence intensity change of the complex capsules ðPSS=PAHÞ5 = ðDMPA þ NBD-DPPCÞ with and without treatment of
L. Ge et al. / Biochemical and Biophysical Research Communications 303 (2003) 653–659
ethanol, we kept the optical parameters of the CLSM constant during the measurements. Five percent NBDDPPC solution was prepared for the detection that phospholipid was adsorbed on the surface of the multilayer polyelectrolyte capsules. Fig. 1A shows the CLSM image of phospholipid covered hollow shells. The green ring with a diameter around 2.85 lm demonstrates that the phospholipids have been adsorbed on the surface of the capsules. The fluorescence intensity distribution from the lipids is very homogeneous. This means that the adsorption of phospholipid to the surface of charged polyelectrolyte capsules is quite uniform. Fig. 1B gives the fluorescence intensity distribution along the line drawn in Fig. 1A. As one can see, the intensity peaks on both walls of the capsule were similar and have a value of about 250 U (the peak value of I and II in Fig. 1B). One can also see that there is intensity above the noise level from inside of the capsules indicating some dye there. This has been verified by using small dye molecules (6-CF) in the previous experiments [14].
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In previous experiments we learned that lipid vesicles and liposomes were unstable towards many surfactants and alcoholic derivatives [21]. If one considers the lipidcoated capsules as a special vesicle, one may find that their properties are similar to those of lipid vesicles. To
show this, we chose dilute ethanol solutions to wash DMPA coated shells and then re-dispersed the capsules into water. The experimental process was described in step 2 of Scheme 1. Fig. 2 shows the CLSM image of ðPSS=PAHÞ5 =DMPA vesicles after exposure to the above dilute ethanol solution. In comparison with Fig. 1B, one can find that the fluorescence intensity on the capsule walls decreased dramatically, indicating that the lipid layers on the surface of the capsules have been damaged. After rinsing, the fluorescence intensity of the rings obviously is reduced, indicating that most of the lipid molecules have been removed from the surface of the capsules, as shown in Fig. 2. Electrophoretic mobility measurements confirmed that as the outermost layer is PAH, the zeta-potential of the surface is positive with a value of þ43:0 0:8 mV. After the mixed DMPA and NBD-DPPC was subsequently adsorbed on the PAH layer, the zeta-potential of the surface is negative with a value of 37:6 0:1 mV. After the complex capsules were treated with alcohol solution, the zetapotential of the surface became positive with a value of þ35:4 0:8 mV, indicating the removal of anionic lipids. Detailed experimental results are displayed in Table 1. Based on the mechanism of layer-by-layer assembly, one can use positively charged PAH to cover the negatively charged surface of the ðPSS=PAHÞ5 =DMPA capsules as described in step 3 of Scheme 1. In fact, by employing rhodamine B labeled PAH (Rb-PAH) to
Fig. 1. (A) CLSM image of a five PSS/PAH layers polyelectrolyte capsule coated with DMPA and 5% NBD-DPPC, showing the lipid vesicles formed on the surface. (B) Profile of the fluorescence intensity along the line in (A).
Fig. 2. (A) CLSM image of ðPSS=PAHÞ5 =DMPA vesicles after exposure to ethanol solution, showing the damage of the lipid vesicles on the shells. (B) The profile diagram of the fluorescence intensity along the line.
Stability of ðPSS=PAH Þ5 =DMPA capsules
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Table 1 Column bar picture of zeta-potential measured for different vesicle surfaces after various treatments
treat the DMPA coated capsules, we observe the red fluorescence rings as shown in Fig. 3, indicating that PAH molecules were adsorbed on the surface of the ðPSS=PAHÞ5 =DMPA capsule. We have proved previously that the polymer can stabilize the lipid vesicles against detergents through electrostatic interaction [19]. In view of this, we added PAH solution to the ðPSS=PAHÞ5 =ðDMPA þ NBD-DPPCÞ suspensions. The final complexes ðPSS=PAHÞ5 =ðDMPA þNBDDPPCÞ=PAH were treated with dilute ethanol and washed out by water (step 4 of Scheme 1). Then we re-dispersed this complex into water. Fig. 4 displays the CLSM image of the ðPSS=PAHÞ5 =DMPA capsules after coating with PAH. The fluorescence intensity on the capsule wall is much stronger than that without PAH protection as shown in Fig. 2 with the same treatment. This means that after PAH adsorption, the desorption of phospholipids was to some extent prevented from the surface of the capsules. As a consequence, PAH stabilizes the ðPSS=PAHÞ5 =ðDMPA þ NBD-DPPCÞ capsules. After the ethanol treatment of the ðPSS=PAHÞ5 =
Fig. 3. CLSM image of ðPSS=PAHÞ5 =DMPA vesicles coated with RbPAH.
Fig. 4. (A) CLSM image of a ðPSS=PAHÞ5 =DMPA=PAH vesicle after exposure to ethanol solution, showing the existence of lipid vesicles protected by the inner and outer polymer layers. (B) Profile of the fluorescence intensity along the line in (A).
ðDMPA þ NBD-DPPCÞ=PAH complex, the zeta-potential was positive with a value of þ39:0 0:6 mV. Permeability control of ðPSS=PAH Þ5 =DMPA complex capsules Pure polyelectrolyte multilayer capsules are easily permeable to 6-CF molecules [15]. We took one drop of ðPSS=PAHÞ5 =DMPA solution and left it on the glass to mix with 6-CF dyes and covered it by a thin glass plate against capsules moving. Then we used the CLSM to measure the permeability of ðPSS=PAHÞ5 =DMPA to 6CF dyes. As one can see in Fig. 5, the inside of the capsules was dark and the green fluorescence was from outside the polyelectrolyte shells. This suggests that the capsules are less impermeable to the small dye molecules. As described above if the ðPSS=PAHÞ5 =DMPA capsules are treated with alcohol solution most of the phospholipid layers would be removed from the surface. Fig. 6 shows the CLSM images of the ðPSS=PAHÞ5 = DMPA capsules after treatment with ethanol. It is found that the dye molecules existed inside and outside the capsules. Therefore, the removal of phospholipid by dilute ethanol solution thus results in an increased permeability of the capsules for 6-CF. We have stated previously that PAH adsorbed on the surface of the ððPSS=PAHÞ5 =DMPAÞ complex capsules via electrostatic interaction stabilizes the vesicles. In Fig. 7A we presented an image of the ðPSS=PAHÞ5 =
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Fig. 5. CLSM image of 6-CF in the ðPSS=PAHÞ5 =DMPA vesicle solution, indicating the impermeability of the ðPSS=PAHÞ5 =DMPA wall to small dye molecules.
Fig. 7. CLSM image of (A) 6-CF dyes added to ðPSS=PAHÞ5 = DMPA=PAH vesicle solution. (B) Then exposed to 10% ethanol solution, showing the stability of ðPSS=PAHÞ5 =DMPA=PAH vesicles to ethanol solution. (C) Exposed to concentrated ethanol solution.
Fig. 6. CLSM image of 6-CF after ðPSS=PAHÞ5 =DMPA vesicles exposure to ethanol solution, showing the penetration of the 6-CF dyes through the vesicle wall.
the penetration of 6-CF. Hence, the polymer can protect the lipid to some extent. In the specific example one observes that the half time for recovery is increased from 10 to 30 s. Diffusion process for dyes across the wall of capsules
DMPA=PAH capsules with 6-CF solution without ethanol treatment. The darkness inside reveals that ðPSS=PAHÞ5 =DMPA=PAH capsules are impermeable to 6-CF dye molecules. Thus, there is no change in permeability after PAH is adsorbed on the surface of ðPSS=PAHÞ5 =DMPA capsules. After the ðPSS=PAHÞ5 =DMPA=PAH capsules were treated with dilute ethanol solution and then mixed with 6-CF solution, CLSM measurements showed that 6-CF dye molecules could not penetrate into the wall of the capsules, the dark centers as shown in Fig. 7B remain in this case, the ðPSS=PAHÞ5 =DMPA=PAH capsules are impermeable to 6-CF. Thus, the PAH as the outermost layer can protect the phospholipid from being removed by dilute ethanol. To testify the permeability limitation of ðPSS= PAHÞ5 =DMPA=PAH to 6-CF, a concentrated ethanol solution was used, as shown in step 5 of Scheme 1, the lipid layers were damaged and 6-CF molecules again could penetrate into the ðPSS=PAHÞ5 =DMPA= PAH capsules. A stronger fluorescence intensity caused by 6CF has been observed inside the capsules as shown in Fig. 7C, which means that a higher concentration of alcohol destroys the lipid layers formed on the ðPSS=PAHÞ5 =DMPA=PAH capsule surface, resulting in
In order to further study the diffusion of dye molecules across the wall of ðPSS=PAHÞ5 =DMPA=PAH capsules, we carried out photo-bleaching experiments. A laser beam was focused on the inner volume of the capsules. After a certain time, most of the dye molecules were bleached. Then recovery was studied by applying lower excitation intensity. A scheme to describe the experimental process is given in Fig. 8A. Fig. 8B shows some images selected from bleach and recovery experiment and Fig. 8C gives the time dependence of the bleach and recovery. In Fig. 8B, as one can see, full bleaching results in the dark field inside of the capsules. Then with time elapsing, the fluorescence intensity from dye molecules inside the capsules is getting stronger. Finally the fluorescence intensity inside and outside the capsules did not change any longer, indicating equilibrium between permeation and bleaching. From the recovery curves of Figs. 8C and D, one can know the recovery time of 6-CF dyes across the wall of ðPSS=PAHÞ5 =DMPA with treatments of dilute ethanol solution and the one of ðPSS=PAHÞ5 = DMPA=PAH which was dispersed into high concentration of EtOH solution. One further verifies that PAH decreases the permeability of the complex capsules for small molecules and protects the lipid in some sense.
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Conclusions A complex capsule system, ðPSS=PAHÞ5 =DMPA has been constructed, which exhibits impermeability for small dye molecule, such as 6-CF. The permeability of the capsules can be changed by ethanol solutions because of dissolution of the lipid membrane.
By using the layer-by-layer technique, the charged polymer PAH can be adsorbed on the surface of ðPSS=PAHÞ5 =DMPA capsules. The complex capsules of ðPSS=PAHÞ5 =DMPA=PAH have a good stability to the dilute ethanol solution. Under treatment with dilute ethanol solution, the lipid-based capsules are stable, indicating that the outermost PAH layer acts as a stabilizer for
Fig. 8. (A) Schematic representation of the photo-bleaching and recovery experiment. (B) Experimental process of photo-bleaching and recovery. (C) Recovery profile for phospholipid coated capsules, treated with EtOH solution then re-dispersed into water. (D) Recovery profile for polymer stabilized phospholipids dispersed into high concentration EtOH.
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the complex capsules. ðPSS=PAHÞ5 = DMPA=PAH capsules are impermeable to small molecules after treatment with ethanol. The photo-bleaching experiments demonstrate that it will take a much longer time to allow small molecules to diffuse into the ðPSS=PAHÞ5 =DMPA=PAH capsules. Hence liposomes can be prepared that are stabilized against organic solvent. The fact that lipid layers adsorbed on the capsule surface can form complex capsules creates a novel route for modeling artificial cells. They have functional features with high stability and well-determined size. The outermost layer coated with polyelectrolytes protects the complexes, which is useful for controlled molecular release applications.
Acknowledgments This research was financially supported by the National Nature Science Foundation of China (NNSFC29925307), the major state basic research development program (973, Grant No. G2000078103) as well as the research contract between the German Max-Planck-Society and the Chinese Academy of Sciences. The authors would also like to acknowledge Christine Pilz for the zeta-potential measurements.
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Polymer-stabilized phospholipid vesicles formed on polyelectrolyte multilayer capsules Liqin Ge,a Helmuth M€ ohwald,b and Junbai Lia,* a
International Joint Lab, The Center for Molecular Science, Institute of Chemistry, Chinese Academy of Science, Beijing 100080, China b Max-Planck-Institute of Colloids and Interfaces, Am Muehlenberg 2, Golm/Potsdam D-14476, Germany Received 18 February 2003
Abstract Phospholipid vesicles on polyelectrolyte multilayer shells can be stabilized against ethanol by coating a single cationic polyelectrolyte. Confocal laser scanning microscopy (CLSM) proved that the lipids were stabilized by cationic polyelectrolytes and the permeability to small hydrophilic dyes was decreased. Measurements of fluorescence recovery after photo-bleaching (FRAP) with individual capsules enable quantification of release profiles. Ó 2003 Published by Elsevier Science (USA). Keywords: Release; Vesicles; Phospholipids; Polyelectrolyte shells; Polymer complex; CLSM
Phospholipid vesicles have received much attention as possible drug carriers and model systems to study biological processes at membranes [1–5]. In many practical applications, however, they lack the stable natural system, which is gained by coating of biopolymers on both sides of the membrane, and which is difficult to realize [3]. Consequently, these attempts to stabilize the bilayer via polymerization were mostly unsuccessful due to lateral phase separation [4–6]. As an alternative, one may prepare vesicles by adsorbing polyelectrolytes on both sides and a variant of this approach has been tested [7,8]. In order to prepare stabilized vesicles with a defined size, an alternative strategy has recently been developed by employing layer-by-layer technique. Polyelectrolyte multilayers were prepared on colloidal particles that were later sacrificed by dissolution to obtain hollow capsules. On these capsules a phospholipid bilayer is adsorbed, thereafter, a polycation layer was coated. In this way, one can obtain vesicles with a defined size, shape, and achieve some stability [9–17]. An understanding of such complex vesicle characteristics is not only of scientific interest but is also motivated by release applications [15,18]. It has been shown * Corresponding author. Fax: +86-10-8261-2484. E-mail address: [email protected] (J. Li).
from our previous studies that polyelectrolytes can stabilize liposomes [19]. Five layer ðPSS=PAHÞ5 capsules are one of the most frequently investigated systems. Thus we try to make use of polyelectrolytes to modify the lipid/capsule complex vesicles. The fabrication procedure and the stabilization mechanisms of the ðPSS=PAHÞ5 =DMPA=PAH vesicles are demonstrated in the following Scheme 1A and B, respectively.
Materials and methods Materials. The sources of chemicals were as follows: poly(styrenesulfonate, sodium salt) (PSS, Mw 70,000) and poly(allylamine hydrochloride) (PAH, Mw 70,000) were from Aldrich. L -a-Dimyristoylphosphatidic acid (DMPA), 6-carboxyfluorescein (6-CF), fluorescently labeled DPPC (L -a-phosphatidylcholine (NBD-aminohexanoyl)-c-palmitoyl) (NBD-DPPC), and ethanol were purchased from Sigma. Two ethanol solutions were prepared with different ratios: VEtOH :Vwater ¼ 1:10 and 1:1. All these materials were used as received. PAH was covalently labeled by rhodamine isothiocyanate (molecular probes) based on [20]. The molecular structures of the compounds used in this study are shown in Scheme 2. The water used in all experiments was prepared in a three-stage Millipore Milli-Q Plus 185 purification system and had a resistivity higher than 18:2 MX cm1 . Positively charged melamine formaldehyde particles (MF particles) with a diameter of around 2:85 0:09 lm were obtained from Microparticles GmbH, Berlin.
0006-291X/03/$ - see front matter Ó 2003 Published by Elsevier Science (USA). doi:10.1016/S0006-291X(03)00391-7
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Scheme 1. Illustration of procedures for preparing the ðPSS=PAHÞ5 =DMPA vesicles with a further coating by polyelectrolytes (A) and the stabilization of test of the complex vesicles (B).
Scheme 2. Molecular structures of the compounds used in this study. Polyelectrolyte shells prepared through layer-by-layer adsorption. Multilayer assembly was accomplished by the adsorption of polyelectrolytes at a concentration of 1 mg/ml, 0.5 M NaCl aqueous solutions. Oppositely charged polyelectrolyte species were subsequently added to the suspension followed by repeated centrifugation cycles. After the intended number of layers was adsorbed, 0.1 M HCl was used to remove the core (MF particles) and the obtained hollow polyelectrolyte shells were obtained [14]. PSS is used to form the first layer and the outermost layer is PAH, which has a positive charge in order to bind the negatively charged phospholipids in the next step. Preparation of phospholipid solution. The phospholipid, DMPA, was dissolved in a mixed solvent of chloroform and methanol (VCHCl3 :VMeOH ¼ 1:1) with a concentration of 0.5 mg/ml. Then the solvent was evaporated in a rotavap at 30 °C, and afterwards water was added to a final lipid concentration (CDMPA ¼ 0:025 mg=ml), and sonicated for 5 min. Lipid solution was added into polyelectrolyte shells and allowed 5 min for adsorption. Then the mixed solution was washed for at least three times with water to remove the non-adsorbed materials by centrifugation.
Measurements of CLSM. The images of capsules were obtained by a Leica confocal scanning system. A 100 oil immersion objective with a numerical aperture of 1.4 was used. In the experiment, we used fluorescently labeled NBD-DPPC at 5 wt%. 6-Carboxyfluorescein (6-CF) and rhodamine b-PAH (Mw 65,000) were selected as hydrophilic fluorescence dyes. Measurements of FRAP. In order to study the penetration of dyes across the wall, the dyes inside the capsules were photo-chemically bleached. To do this, an argon ion laser from the CLSM emitting at a wavelength of k ¼ 488 nm was used. The laser beam was focused onto selected area inside the capsule. The time for bleaching was long enough to ensure that almost all dye molecules in the capsule were bleached. Imaging was typically performed at a rather low laser intensity. The interval between each image scan varied with the duration of recovery established at an initial pilot experiment. Recovery was considered complete when the intensity of the photo-bleached region was stable and the curve was flat. For quantitative analysis, the fluorescence intensity was integrated by tracing a fixed area in the interior (ROI analysis system provided by the CLSM software), giving an intensity value for each time point. We have to point out here that there is always a small amount of fluorescence dyes lost during repeatedly scanning and recovering. However, this will not influence the experimental results. Electrophoretic mobility measurements. The zeta-potential of the capsules was measured by a Malvern Zetasizer 4. The mobility l was converted into a zeta (f)-potential by the Smoluchowski relation, f ¼ lg=e, where g and e are the viscosity and permeability of the solution, respectively [14].
Results and discussion Assembly of phospholipids vesicles onto polyelectrolyte capsules To compare quantitatively the fluorescence intensity change of the complex capsules ðPSS=PAHÞ5 = ðDMPA þ NBD-DPPCÞ with and without treatment of
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ethanol, we kept the optical parameters of the CLSM constant during the measurements. Five percent NBDDPPC solution was prepared for the detection that phospholipid was adsorbed on the surface of the multilayer polyelectrolyte capsules. Fig. 1A shows the CLSM image of phospholipid covered hollow shells. The green ring with a diameter around 2.85 lm demonstrates that the phospholipids have been adsorbed on the surface of the capsules. The fluorescence intensity distribution from the lipids is very homogeneous. This means that the adsorption of phospholipid to the surface of charged polyelectrolyte capsules is quite uniform. Fig. 1B gives the fluorescence intensity distribution along the line drawn in Fig. 1A. As one can see, the intensity peaks on both walls of the capsule were similar and have a value of about 250 U (the peak value of I and II in Fig. 1B). One can also see that there is intensity above the noise level from inside of the capsules indicating some dye there. This has been verified by using small dye molecules (6-CF) in the previous experiments [14].
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In previous experiments we learned that lipid vesicles and liposomes were unstable towards many surfactants and alcoholic derivatives [21]. If one considers the lipidcoated capsules as a special vesicle, one may find that their properties are similar to those of lipid vesicles. To
show this, we chose dilute ethanol solutions to wash DMPA coated shells and then re-dispersed the capsules into water. The experimental process was described in step 2 of Scheme 1. Fig. 2 shows the CLSM image of ðPSS=PAHÞ5 =DMPA vesicles after exposure to the above dilute ethanol solution. In comparison with Fig. 1B, one can find that the fluorescence intensity on the capsule walls decreased dramatically, indicating that the lipid layers on the surface of the capsules have been damaged. After rinsing, the fluorescence intensity of the rings obviously is reduced, indicating that most of the lipid molecules have been removed from the surface of the capsules, as shown in Fig. 2. Electrophoretic mobility measurements confirmed that as the outermost layer is PAH, the zeta-potential of the surface is positive with a value of þ43:0 0:8 mV. After the mixed DMPA and NBD-DPPC was subsequently adsorbed on the PAH layer, the zeta-potential of the surface is negative with a value of 37:6 0:1 mV. After the complex capsules were treated with alcohol solution, the zetapotential of the surface became positive with a value of þ35:4 0:8 mV, indicating the removal of anionic lipids. Detailed experimental results are displayed in Table 1. Based on the mechanism of layer-by-layer assembly, one can use positively charged PAH to cover the negatively charged surface of the ðPSS=PAHÞ5 =DMPA capsules as described in step 3 of Scheme 1. In fact, by employing rhodamine B labeled PAH (Rb-PAH) to
Fig. 1. (A) CLSM image of a five PSS/PAH layers polyelectrolyte capsule coated with DMPA and 5% NBD-DPPC, showing the lipid vesicles formed on the surface. (B) Profile of the fluorescence intensity along the line in (A).
Fig. 2. (A) CLSM image of ðPSS=PAHÞ5 =DMPA vesicles after exposure to ethanol solution, showing the damage of the lipid vesicles on the shells. (B) The profile diagram of the fluorescence intensity along the line.
Stability of ðPSS=PAH Þ5 =DMPA capsules
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Table 1 Column bar picture of zeta-potential measured for different vesicle surfaces after various treatments
treat the DMPA coated capsules, we observe the red fluorescence rings as shown in Fig. 3, indicating that PAH molecules were adsorbed on the surface of the ðPSS=PAHÞ5 =DMPA capsule. We have proved previously that the polymer can stabilize the lipid vesicles against detergents through electrostatic interaction [19]. In view of this, we added PAH solution to the ðPSS=PAHÞ5 =ðDMPA þ NBD-DPPCÞ suspensions. The final complexes ðPSS=PAHÞ5 =ðDMPA þNBDDPPCÞ=PAH were treated with dilute ethanol and washed out by water (step 4 of Scheme 1). Then we re-dispersed this complex into water. Fig. 4 displays the CLSM image of the ðPSS=PAHÞ5 =DMPA capsules after coating with PAH. The fluorescence intensity on the capsule wall is much stronger than that without PAH protection as shown in Fig. 2 with the same treatment. This means that after PAH adsorption, the desorption of phospholipids was to some extent prevented from the surface of the capsules. As a consequence, PAH stabilizes the ðPSS=PAHÞ5 =ðDMPA þ NBD-DPPCÞ capsules. After the ethanol treatment of the ðPSS=PAHÞ5 =
Fig. 3. CLSM image of ðPSS=PAHÞ5 =DMPA vesicles coated with RbPAH.
Fig. 4. (A) CLSM image of a ðPSS=PAHÞ5 =DMPA=PAH vesicle after exposure to ethanol solution, showing the existence of lipid vesicles protected by the inner and outer polymer layers. (B) Profile of the fluorescence intensity along the line in (A).
ðDMPA þ NBD-DPPCÞ=PAH complex, the zeta-potential was positive with a value of þ39:0 0:6 mV. Permeability control of ðPSS=PAH Þ5 =DMPA complex capsules Pure polyelectrolyte multilayer capsules are easily permeable to 6-CF molecules [15]. We took one drop of ðPSS=PAHÞ5 =DMPA solution and left it on the glass to mix with 6-CF dyes and covered it by a thin glass plate against capsules moving. Then we used the CLSM to measure the permeability of ðPSS=PAHÞ5 =DMPA to 6CF dyes. As one can see in Fig. 5, the inside of the capsules was dark and the green fluorescence was from outside the polyelectrolyte shells. This suggests that the capsules are less impermeable to the small dye molecules. As described above if the ðPSS=PAHÞ5 =DMPA capsules are treated with alcohol solution most of the phospholipid layers would be removed from the surface. Fig. 6 shows the CLSM images of the ðPSS=PAHÞ5 = DMPA capsules after treatment with ethanol. It is found that the dye molecules existed inside and outside the capsules. Therefore, the removal of phospholipid by dilute ethanol solution thus results in an increased permeability of the capsules for 6-CF. We have stated previously that PAH adsorbed on the surface of the ððPSS=PAHÞ5 =DMPAÞ complex capsules via electrostatic interaction stabilizes the vesicles. In Fig. 7A we presented an image of the ðPSS=PAHÞ5 =
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Fig. 5. CLSM image of 6-CF in the ðPSS=PAHÞ5 =DMPA vesicle solution, indicating the impermeability of the ðPSS=PAHÞ5 =DMPA wall to small dye molecules.
Fig. 7. CLSM image of (A) 6-CF dyes added to ðPSS=PAHÞ5 = DMPA=PAH vesicle solution. (B) Then exposed to 10% ethanol solution, showing the stability of ðPSS=PAHÞ5 =DMPA=PAH vesicles to ethanol solution. (C) Exposed to concentrated ethanol solution.
Fig. 6. CLSM image of 6-CF after ðPSS=PAHÞ5 =DMPA vesicles exposure to ethanol solution, showing the penetration of the 6-CF dyes through the vesicle wall.
the penetration of 6-CF. Hence, the polymer can protect the lipid to some extent. In the specific example one observes that the half time for recovery is increased from 10 to 30 s. Diffusion process for dyes across the wall of capsules
DMPA=PAH capsules with 6-CF solution without ethanol treatment. The darkness inside reveals that ðPSS=PAHÞ5 =DMPA=PAH capsules are impermeable to 6-CF dye molecules. Thus, there is no change in permeability after PAH is adsorbed on the surface of ðPSS=PAHÞ5 =DMPA capsules. After the ðPSS=PAHÞ5 =DMPA=PAH capsules were treated with dilute ethanol solution and then mixed with 6-CF solution, CLSM measurements showed that 6-CF dye molecules could not penetrate into the wall of the capsules, the dark centers as shown in Fig. 7B remain in this case, the ðPSS=PAHÞ5 =DMPA=PAH capsules are impermeable to 6-CF. Thus, the PAH as the outermost layer can protect the phospholipid from being removed by dilute ethanol. To testify the permeability limitation of ðPSS= PAHÞ5 =DMPA=PAH to 6-CF, a concentrated ethanol solution was used, as shown in step 5 of Scheme 1, the lipid layers were damaged and 6-CF molecules again could penetrate into the ðPSS=PAHÞ5 =DMPA= PAH capsules. A stronger fluorescence intensity caused by 6CF has been observed inside the capsules as shown in Fig. 7C, which means that a higher concentration of alcohol destroys the lipid layers formed on the ðPSS=PAHÞ5 =DMPA=PAH capsule surface, resulting in
In order to further study the diffusion of dye molecules across the wall of ðPSS=PAHÞ5 =DMPA=PAH capsules, we carried out photo-bleaching experiments. A laser beam was focused on the inner volume of the capsules. After a certain time, most of the dye molecules were bleached. Then recovery was studied by applying lower excitation intensity. A scheme to describe the experimental process is given in Fig. 8A. Fig. 8B shows some images selected from bleach and recovery experiment and Fig. 8C gives the time dependence of the bleach and recovery. In Fig. 8B, as one can see, full bleaching results in the dark field inside of the capsules. Then with time elapsing, the fluorescence intensity from dye molecules inside the capsules is getting stronger. Finally the fluorescence intensity inside and outside the capsules did not change any longer, indicating equilibrium between permeation and bleaching. From the recovery curves of Figs. 8C and D, one can know the recovery time of 6-CF dyes across the wall of ðPSS=PAHÞ5 =DMPA with treatments of dilute ethanol solution and the one of ðPSS=PAHÞ5 = DMPA=PAH which was dispersed into high concentration of EtOH solution. One further verifies that PAH decreases the permeability of the complex capsules for small molecules and protects the lipid in some sense.
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Conclusions A complex capsule system, ðPSS=PAHÞ5 =DMPA has been constructed, which exhibits impermeability for small dye molecule, such as 6-CF. The permeability of the capsules can be changed by ethanol solutions because of dissolution of the lipid membrane.
By using the layer-by-layer technique, the charged polymer PAH can be adsorbed on the surface of ðPSS=PAHÞ5 =DMPA capsules. The complex capsules of ðPSS=PAHÞ5 =DMPA=PAH have a good stability to the dilute ethanol solution. Under treatment with dilute ethanol solution, the lipid-based capsules are stable, indicating that the outermost PAH layer acts as a stabilizer for
Fig. 8. (A) Schematic representation of the photo-bleaching and recovery experiment. (B) Experimental process of photo-bleaching and recovery. (C) Recovery profile for phospholipid coated capsules, treated with EtOH solution then re-dispersed into water. (D) Recovery profile for polymer stabilized phospholipids dispersed into high concentration EtOH.
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the complex capsules. ðPSS=PAHÞ5 = DMPA=PAH capsules are impermeable to small molecules after treatment with ethanol. The photo-bleaching experiments demonstrate that it will take a much longer time to allow small molecules to diffuse into the ðPSS=PAHÞ5 =DMPA=PAH capsules. Hence liposomes can be prepared that are stabilized against organic solvent. The fact that lipid layers adsorbed on the capsule surface can form complex capsules creates a novel route for modeling artificial cells. They have functional features with high stability and well-determined size. The outermost layer coated with polyelectrolytes protects the complexes, which is useful for controlled molecular release applications.
Acknowledgments This research was financially supported by the National Nature Science Foundation of China (NNSFC29925307), the major state basic research development program (973, Grant No. G2000078103) as well as the research contract between the German Max-Planck-Society and the Chinese Academy of Sciences. The authors would also like to acknowledge Christine Pilz for the zeta-potential measurements.
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