Composition and Properties of Complexes between Spherical

Nov 2, 2012 - Andrey V Sybachin , Polina O Khlynina , Vasiliy V Spiridonov , Irina G .... V. Zaborova , Viktor N. Orlov , Matthias Ballauff , Yeshayah...
20 downloads 0 Views 2MB Size
Download PDF
Article pubs.acs.org/Langmuir

Composition and Properties of Complexes between Spherical Polycationic Brushes and Anionic Liposomes Andrey V. Sybachin,*,† Olga V. Zaborova,† Matthias Ballauff,‡,§ Ellina Kesselman,∥ Judith Schmidt,∥ Yeshayahu Talmon,∥ Fredric M. Menger,⊥ and Alexander A. Yaroslavov† †

Department of Chemistry, M.V. Lomonosov Moscow State University, Leninskie Gory 1-3, 119991 Moscow, Russian Federation Physikalische Chemie I, Universität Bayreuth, 95440 Bayreuth, Germany § Helmholtz-Zentrum Berlin für Materialien und Energie, Hahn-Meitner-Platz 1, D-14109 Berlin, Germany ∥ Department of Chemical Engineering, Technion-Israel Institute of Technology, 32000 Haifa, Israel ⊥ Department of Chemistry, Emory University, Atlanta, Georgia 30322, United States ‡

ABSTRACT: A spherical polycationic brush (SPB) is made by graft-polymerizing a cationic monomer onto the surface of a 100 nm polystyrene bead. It is possible to adsorb anionic liposomes (40−60 nm diameter) onto the SPBs while maintaining the liposome integrity. The liposomes were constructed with phosphatidyl choline (PC) admixed with 0.05−0.4 mol fraction of an dianionic lipid, cardiolipin (CL2−). As shown by electrophoretic mobility measurements, SPB-to-liposome complexation leads to a conversion from the initial positive charge of the copolymer to a negative charge. The higher the CL2− content of the liposomes, the lower the concentration needed for charge neutralization. Dynamic light scattering (DLS) revealed that multicomplex aggregates are formed with a maximum size at the SPB/liposome charge-equivalence point. Experiments with fluorescent-labeled liposomes show that at low CL2− content about 80 liposomes are adsorbed per SPB. As the mole fraction of CL2− increases from 0.05 to 0.4, fewer liposomes adsorb owing to electrostatic repulsion among neighboring liposomes. The effect of added NaCl also depends upon the CL2− content. With 0.05 mol fraction CL2−, the SPB/liposome complex dissociates into its components at 0.15 M NaCl. With a mole fraction of >0.1, complexes fail to dissociate even at 1.2 M NaCl. Additional information about the SPB/liposome morphology was obtained from cryo-TEM. For example, cryo-TEM data confirm liposome integrity upon complexation, a behavior that contrasts with the liposome destruction as found with adsorption to many other types of surfaces.

■

INTRODUCTION Use of spherical bilayer lipid vesicles (liposomes) for encapsulation and release of small bioactive compounds was suggested immediately following their preparation and description in the middle of last century.1 Due to their unique structure, liposomes have been shown to entrap both hydrophilic and hydrophobic guests: the former dissolve within the internal water pool, whereas the latter enter the lipid bilayer.2,3 In spite of considerable progress in liposomal containers, difficulties were encountered4 owing to their low thermodynamic stability;5 their high uptake by reticularendothelial cells;6 their relatively small capture volume;7 and problems with their vectorial modification.8 Experiments, undertaken to immobilize liposomal containers on the surface of solid carriers (implants), led in most cases to liposome disruption and uncontrolled release of entrapped drugs.9 Successful methods of liposome immobilization require the use of special preliminary procedures for modifying both the liposomes and the adsorbent surface.10 Recently, we described a method of anionic liposome adsorption on the surface of colloidal particles covered by grafted polycationic chains (“spherical polycationic brushes”, SPB) that maintained liposome integrity.11 In the present © XXXX American Chemical Society

article, we describe complexation of cationic SPBs and anionic liposomes with varying content of anionic lipid. The capacity of SPBs toward anionic liposomes, the stability of resulting complexes in aqueous salt solutions, and the nanostructure of the complexes as shown by cryo-TEM were also examined.

■

EXPERIMENTAL SECTION

SPBs were synthesized by graft polymerization of a cationic monomer, (2-methylpropenoyloxyethyl)trimethylammonium chloride, on the surface of monodispersed polystyrene (PS) latex particles ca. 100 nm in diameter.12 With the aid of dynamic light scattering a mean hydrodynamic diameters of the brushes was found to be 230 nm, that gave a thickness of a cationic corona (a contour length, Lc) equal to (230−100)/2 = 65 nm. The SPB structure is schematically presented in Figure 1. SPB concentration ([SPB+]) is expressed in moles of cationic subunit per liter. Zwitterionic phosphatidylcholine (PC) (I), dual anionic diphosphatidylglycerol (cardiolipin, CL2−) (II), and Nfluorescein-isothiocyanyldipalmitoyl-phosphatidylethanolamine (FITC-DPPE) (III) from Avanti were used as received (Figure 2). Received: June 14, 2012 Revised: October 4, 2012

A

dx.doi.org/10.1021/la3024265 | Langmuir XXXX, XXX, XXX−XXX

Langmuir

Article

The fluorescence intensity of FITC-labeled liposome suspensions was measured at λem = 525 nm (λex = 495 nm) using a F-4000 Hitachi fluorescence spectrofluorimeter. UV−vis spectra of labeled liposomes were measured with a UV-mini 1240 Hitachi spectrophotometer. Mean hydrodynamic diameters of SPBs, liposomes, and SPB/ liposome complexes were determined by dynamic light scattering at the fixed scattering angle (90°) in a thermostatic cell with a Brookhaven Zeta Plus instrument. Software provided by the manufacturer was employed to calculate diameter values. The size of PC/CL2− liposomes was found to fluctuate from sample to sample but always fell into the 40−60 nm range with a mean value of 50 nm. No systematic change in the liposome size with CL2− content (ν) was detected. Electrophoretic mobility (EPM) of SPBs, liposomes, and SPB/liposome complexes was measured by laser microelectrophoresis in a thermostatic cell using a Brookhaven Zeta Plus instrument with the corresponding software. Permeability of the liposomal membranes toward a simple salt was investigated by measuring the conductivity of NaCl-loaded vesicle suspensions with a CDM83 conductometer (Radiometer) as described in ref 11. In all experiments, the adsorption phenomena were completed within a few seconds after mixing of the components. This is demonstrated, for example, by time-dependent fluorescence changes when mixing fluorescent liposomes with the brushes. Size changes were slight in the five minutes required to complete electrophoretic, light scattering, and fluorescent experiments. Conductometric changes, resulting exclusively from a release of small ions by the liposomes, are not dependent upon size of SPB/liposome complex particles. Vitrified specimens for cryogenic transmission electron microscopy (cryo-TEM) were prepared in a controlled environment vitrification system (CEVS), where desirable temperature and humidity were

Figure 1. Polycationic brush (schematical presentation). Small unilamellar anionic liposomes were prepared by the standard sonication procedure: appropriate amounts of PC and CL2− solutions in methanol were mixed in a flask, after which the solvent was evaporated under vacuum. The resulting thin lipid film was then dispersed in a borate buffer (pH 9.2, 10−2 M) for 400s with a 4700 Cole-Parmer ultrasonic homogenizer. Liposome samples were separated from titanium dust by centrifugation for 5 min at 10 000 rpm and used within one day. Liposomes with a molar fraction of anionic CL2− head-groups ν = 2[CL2−]/(2[CL2−] + [PC]) from 0.05 up to 0.4 were thus obtained; liposomes with a fluorescent dye incorporated into the membrane were prepared by the same procedure, except that 1 wt % of FITC-DPPE was added to the lipid mixture solution before methanol evaporation.

Figure 2. Lipids (schematical presentation). B

dx.doi.org/10.1021/la3024265 | Langmuir XXXX, XXX, XXX−XXX

Langmuir

Article

maintained. Briefly, a drop of the liposome suspension, or SPB suspension, or mixed SPB/liposome suspension, was placed on a perforated carbon film-coated copper grid, blotted with a filter paper, and plunged into liquid ethane at its freezing point. The vitrified specimens were transferred to an Oxford CT-3500 cooling holder and observed in either a Philips CM120 or an FEI T12 transmission electron microscope at about −180 °C in the low-dose imaging mode to minimize electron-beam radiation damage. Images were digitally recorded with a Gatan 791 MultiScan cooled-CCD camera (CM120) or by a Gatan US1000 high-resolution cooled-CCD camera (T12). Details may be found elsewhere.13,14 Solutions were prepared with double-distilled water that was additionally treated by a Milli-Q Millipore system composed of ionexchange and adsorption columns as well as a filter to remove large particles. Brush-to-liposome binding was examined in 10−2 M borate buffer at 20 °C. Under these conditions, the membranes of PC/CL2− liposomes were in the fluid (liquid-crystalline) state.15

negative) means a more stable colloidal system. A similar behavior was reasonably expected for cationic SPBs as their charges were saturated by anionic liposomes. Actually, the size of SPB/liposome complex particles, determined by dynamic light scattering (Figure 4), showed that the individual

■

RESULTS AND DISCUSSION This paper deals with the interaction between cationic SPBs (Figure 1) and anionic liposomes composed of phosphatidylcholine (PC) and cardiolipin (CL2−) shown in Figure 2. Interparticle binding was accompanied by neutralization of the surface charge of the SPBs as detected by their altered electrophoretic mobility (EPM). Figure 3 shows how the EPM

Figure 4. Hydrodynamic diameter of SPB particles vs PC/CL2− liposome concentration. ν = 0.05 (1), 0.1 (2), 0.2 (3), 0.3 (4), and 0.4 (5). [SPB+] = 10−4 M.

complexes formed multicomplex aggregates with maximum size at EPM = 0 (cf. Figure 4 and Figure 3), but both positive and negative charges on the individual complexes inhibited the aggregation. To estimate the number of liposomes bound to SPBs, the following experiment was carried out. A suspension of SPBs was mixed with a suspension of fluorescent-labeled FITCDPPE liposomes and 5 min after a SPB/liposome complex was separated by centrifugation. The fluorescence intensity in the supernatant was recalculated in the concentration of unbound liposomes using the corresponding calibration curve. A dependence of unbound liposome vs total liposome concentration is given in Figure 5. It is seen that all added liposomes were complexed with to SPBs up to a certain concentration (specific for each ν value); at higher concentrations, free (unbound) liposomes could be found in the supernatant. The data from Figure 5 allowed the calculation of an ultimate liposome number capable of complexing with a single SPB particle11 as

Figure 3. EPM of SPB particles vs PC/CL2− liposome concentration. ν = 0.05 (1), 0.1 (2), 0.2 (3), 0.3 (4), and 0.4 (5). [SPB+] = 10−4 M.

varies with liposomal lipid concentration as the molar fraction of the CL2− relative to the PC (a parameter designated ν) varied from 0.05 up to 0.4. By increasing the CL2− content of the liposomes, the liposomes took on an increasingly negative charge. In all cases, the SPB-to-liposome complexation led to a decrease in the SPB surface charge and an overall change from positive to negative charge at high liposome concentrations. Elevation of the CL2− content required a lower concentration of liposomes for SPB charge neutralization, and at the same time, the greater the CL2− content at excess CL2− (level portion of Figure 3) the lower the negative charge on the SPBs. Charge neutralization (EPM = 0) occurred at 0.86 mg/mL lipid for ν = 0.05 liposomes with charge saturation at EPM = −0.2 (μm/s)/ (V/cm). In contrast, charge neutralization occurred at only 0.27 mg/mL lipid for ν = 0.4 liposomes with charge saturation at EPM = −2.9 (μm/s)/(V/cm). The aggregation stability of hydrophilic colloids is determined, among other factors, by a total surface charge of colloidal particles.16 A higher surface charge (positive or

N = (C lip × S1 × Na × D3 × ρ)/(6C brush × d 2 × M ) (1)

where Clip is lipid concentration at saturation, Cbrush is SPB concentration, D is diameter of polystyrene core of SPB particle and ρ is its density,17 d is mean liposome diameter, 50 nm, S1 is mean surface area per one lipid molecule, 0.7 nm2,15 M is mean molecular weight of the lipid,15 and Na is Avogadro’s number. The calculations based on eq 1 for liposomes with different ν are represented in Figure 6 (curve 1). The N vs ν dependence is nonlinear: N value decreased from ∼80 down to ∼40 when raising ν from 0.05 to 0.1, and became equal to 15 at ν = 0.4. At first thought, we might expect a linear relationship between the ultimate amount of complexed liposomes and the content of anionic lipid within the liposomal membrane. We described above a quantitative binding of liposomes with SPBs (Figure 5). Neutralization of the SPB charge should be linearly C

dx.doi.org/10.1021/la3024265 | Langmuir XXXX, XXX, XXX−XXX

Langmuir

Article

Figure 5. Concentration of PC/CL2− liposomes in supernatant after separation of SPB/liposome complex vs total liposome concentration. ν = 0.05 (1), 0.1 (2), 0.2 (3), 0.3 (4), and 0.4 (5). [SPB+] = 10−4 M.

charge-neutrality on the SPB surface would be achieved. Complexation of anionic liposomes with linear cationic polymers, not attached to solid particle surfaces, follows this mechanism.18 However, our experiments suggest a different behavior of the SPB/liposome complex. A reasonable explanation of the behavior of our SPB/ liposome complexes could be as follows. The membrane of PC/CL2− liposomes is in the liquid-crystalline state where lipid molecules are able to move in lateral and transmembrane directions. Binding of PC/CL2− liposomes to a polycation was shown to induce structural rearrangements within the liposomal membrane, i.e., lateral segregation and transmembrane migration (flip-flop) of anionic lipids. Owing to the presence of the cationic polymer on the external liposome surface, anionic lipids from both membrane leaflets concentrate on the outer leaflet. This has been demonstrated conclusively in previous papers.19−21 Clusters of anionic lipids form as a result of ionic contacts between anionic lipids and cationic polymer units. It is reasonable to assume the similar structural rearrangements in the membrane of PC/CL2− liposomes upon their binding with flexible poly(2-methylpropenoyloxyethyl) trimethylammonium chloride moieties grafted to PS particles.

Figure 6. Number of PC/CL2− liposomes ultimately adsorbed on the surface of a single SPB particle as a function of ν.

dependent on the amount of bound anionic lipids. At a given negative charge, the same for all SPB/liposome complexes,

Figure 7. Complexes of SPBs with EL/CL2− liposomes (schematic presentation). ν = 0.1 (a), 0.3 (b), and 0.4 (c). D

dx.doi.org/10.1021/la3024265 | Langmuir XXXX, XXX, XXX−XXX

Langmuir

Article

Figure 7 shows a schematic presentation of the SPB/ liposome complex structure. The mean hydrodynamic diameter of liposomes was found to lie within 40−60 nm, a distance that exceeds the mean distance between the polycationic chains as grafted onto the polystyrene beads (∼20 nm12). Because of many positive charges, each polycationic chain feels a strong electrostatic repulsion from neighboring chains that results in a compacted and rigid network within the polycationic corona.22,23 Small liposomes, in turn, tend to retain their spherical shape.24,25 This allows a reasonable assumption that the 40−60 nm PC/CL2− liposomes adsorb primarily at the polycation/water interface (on the “top of polycation fingers”), although their partial penetration into the grafted polycation layer cannot be precluded. At a low CL2− content, nearly all CL2− molecules concentrate on the liposome side directly in contact with polycation chains. Only a negligible number of CL2− molecules remain on the opposite, inner leaflet of the adsorbed liposome (Figure 7a). It is these outer CL2− molecules that result in a low total positive charge of SPB/ liposome complex at saturation of the liposomal surface with liposomes (Figure 3, curve 1). This, in turn, allows the considerable number of liposomes (∼80) to be adsorbed on the SPB surface (Figure 6). As the ν value increasingly increases, more CL2− molecules remain on the outer sides of adsorbed liposomes (Figure 7b,c), a fact that ensures the change of the surface charge from positive to negative at saturation (Figure 3, curves 2−5) and a decrease in the number of adsorbed liposomes (Figure 6). There is, therefore, a close connection between the CL2− content in the liposomal membrane, on one hand, and the number of adsorbed liposomes, on the other (Figure 6). An increase in the ν value from 0.05 up to 0.3 is accompanied by a progressive increase in the negative surface charge (Figure 3) and the interliposomal repulsion that, in turn, results in a decrease in the number of adsorbed liposomes (Figure 6). Increase of ν from 0.3 to 0.4 has only a small effect on the surface charge, and only a slight change in the number of adsorbed liposomes is detected (Figure 6). A key question concerns the stability of SPB/liposome complexes in aqueous salt media. Is there dissociation of the complexes in the presence of salts, and if so, at what salt concentration does this occur? An answer to this question is based on the fact that cationic polymers are known to be effective fluorescence quenchers.26 Formation and dissociation of SPB/liposome complexes was controlled by measuring the fluorescence intensity of an FITC-labeled lipid incorporated into the liposomal membrane. Addition of a SPB suspension to a suspension of fluorescent-labeled EL/CL2− liposomes with a particular ν value causes the fluorescence intensity to decrease (Figure 8). Further addition of a NaCl solution to the complex suspension had different effects depending on the anionic lipid content of the adsorbed liposomes (Figure 9). The complexes formed by liposomes with ν = 0.05 dissociated into the individual components (SPBs and liposomes) at [NaCl] = 0.15 M. For complexes of liposomes with ν = 0.1, dissociation became measurable only at [NaCl] = 0.1 M and completed at [NaCl] = 0.25 M. Complexes of liposomes with higher ν failed to dissociate at NaCl concentration up to 1.2 M. Thus, anionic liposomes with ν ≥ 0.2 were irreversibly bound to the polycationic brushes. Irreversible complexation of anionic liposomes with linear polycations can result from incorporation of polycation fragments into the liposomal membrane.27 Such incorporation is often accompanied by formation of defects in the lipid

Figure 8. Relative fluorescence intensity of labeled PC/CL2‑ liposomes vs SPB concentration. ν = 0.05 (1), 0.1 (2), 0.2 (3), 0.3 (4), and 0.4 (5). Total lipid concentration 1 mg/mL.

Figure 9. Changes in the relative fluorescence intensity of labeled PC/ CL2− liposomes complexed with SPB after NaCl addition. ν = 0.05 (1), 0.1 (2), 0.2 (3), 0.3 (4), and 0.4 (5). [SPB+] = 10−4 M; total lipid concentration 1 mg/mL.

packing and a release of water-soluble content from inside liposomes. The integrity of PC/CL2− liposomes complexed with SPBs was analyzed by conductometry. Suspensions of liposomes loaded with a 1 M NaCl solution were prepared. Release of NaCl from liposomes into surrounding solution was accompanied by an increase in the suspension conductivity. The results were compared with the conductivity of a suspension of NaCl-loaded liposomes that had been completely destroyed in the presence of excess surfactant (Triton X-100) and taken as a unit activity. It was found that the conductivity does not change when SPBs were added to liposomes with ν ≤ 0.3 (Figure 10, curves 1−4) but increased in time when SPBs were bound to liposomes with ν = 0.4 (curve 5). In other words, complexaion with SPBs had no effect on the permeability of PC/CL2− liposomes with ν ≤ 0.3 although formation of defects in the membrane of PC/CL2− liposomes were observed at ν = 0.4. Additional information about the morphology of the SPB/ liposome complexes was obtained by cryo-TEM. In these experiments, sonicated EL/CL2− liposomes, 50−200 nm in diameter, were used to facilitate their visualization. Figure 11 E

dx.doi.org/10.1021/la3024265 | Langmuir XXXX, XXX, XXX−XXX

Langmuir

Article

Table 1. Characteristics of SPB/Anionic Liposome Complexes ν

N

α

NaCl conc. for complex dissociation, M

integrity of complexed liposomes

0.05 0.1 0.2 0.3 0.4

84 36 20 16 14

1.0 0.46 0.25 0.20 0.18

0.13 0.25 >1.2 >1.2 >1.2

yes yes yes yes no (?)

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

Figure 10. Time-dependent changes in the relative conductivity of a SPB/liposome suspension. ν = 0.05 (1), 0.1 (2), 0.2 (3), 0.3 (4), and 0.4 (5). [SPB+] = 10−4 M; total lipid concentration 1 mg/mL.

ACKNOWLEDGMENTS This work was supported by Russian Foundation for Fundamental Research (projects 11-03-92487; 11-03-00936) and Ministry of Science and Technology of Israel.

■

REFERENCES

(1) Bangham, A. D. Physical structure and behavior of lipids and lipid enzymes. Adv. Lipid Res. 1963, 1, 65. (2) Nii, T.; Ishii, F. Encapsulation efficiency of water-soluble and insoluble drugs in liposomes prepared by the microencapsulation vesicle method. Int. J. Pharm. 2005, 298, 198. (3) Thompson, A. K.; Couchoud, A.; Singh, H. Comparison of hydrophobic and hydrophilic encapsulation using liposomes prepared from milk fat globule-derived phospholipids and soya phospholipids. Dairy Sci. Technol. 2009, 89, 99. (4) Zhang, L.; Gu, F. X.; Chan, J. M.; Wang, A. Z.; Langer, R. S.; Farokhzad, O. C. Nanoparticles in medicine: therapeutic applications and developments. J. Clin. Pharm. Ther. 2008, 83, 761. (5) Lasic, D. D. Liposomes. In From physics to applications; Amsterdam: Elsevier, 1993. (6) Gabizon, A. A.; Shmeeda, H.; Zalipsky, S. Pros and cons of the liposome platform in cancer drug targeting. J. Liposome Res. 2006, 16, 175. (7) Patil, S. G.; Gattani, S. G.; Gaud, R. S.; Surana, S. J.; Dewani, S. P.; Mahajan, H. S. Preparation of liposomes. Pharma Rev. 2005, 18, 53. (8) Torchilin, V. P. Recent advances with liposomes as pharmaceutical carriers. Nat. Rev. Drug Discovery 2005, 4, 145. (9) Richter, R. P.; Bérat, R.; Brisson, A. R. Formation of solidsupported lipid bilayers: An integrated view. Langmuir 2006, 22, 3497. (10) Yoshina-Ishii, C.; Miller, G. P.; Kraft, M. L.; Kool, E. T.; Boxer, S. G. General method for modification of liposomes for encoded assembly on supported bilayers. J. Am. Chem. Soc. 2005, 127, 1356. (11) Yaroslavov, A. A.; Sybachin, A. V.; Schrinner, M.; Ballauff, M.; Tsarkova, L.; Kesselman, E.; Schmidt, J.; Talmon, Y.; Menger, F. M. Liposomes remain intact when complexed with polycationic brushes. J. Am. Chem. Soc. 2010, 132, 5948. (12) Mei, Y.; Wittemann, A.; Sharma, G.; Ballauff, M.; Koch, Th.; Gliemann, H.; Horbach, J.; Schimmel, Th. Engineering the interaction of latex spheres with charged surfaces: AFM investigation of spherical polyelectrolyte brushes on mica. Macromolecules 2003, 36, 3452. (13) Bellare, J. R.; Davis, H. T.; Scriven, L. E.; Talmon, Y. Controlled environment vitrification system: an improved sample preparation technique. J. Electron Microsc. Technol. 1988, 10, 87. (14) Talmon, Y. In Giant micelles, Zana, R., Kaler, E. A., Eds.; CRC Press: New York, 2007; p 163. (15) Torchilin, V., Weissig, V., Eds. Liposomes: A practical approach, 2nd ed., Oxford University Press: New York, 2003. (16) Birdi, K. S., Ed. Surface and colloid chemistry, 2nd ed.; CRC Press: Boca Raton, 2009.

Figure 11. Cryogenic transmission electron microscopy images of mixed SPB+PC/CL2− liposome suspensions. ν = 0.1 and [SPB+] = 2 × 10−4 M (a), ν = 0.3 and [SPB+] = 6 × 10−4 M (b), and ν = 0.4 and [SPB+] = 8 × 10−4 M (c); 0.1 mg/mL lipid concentration.

displays typical cryo-TEM micrographs for complexes formed by liposomes with ν equal to 0.1 (a), 0.3 (b) and 0.4 (c). In all micrographs, there are SPBs (black disks) surrounded by spherical (undisrupted) liposomes (black circles). The cryoTEM data thus corroborate the integrity of liposomes with ν ≤ 0.3 after their binding to SPBs as demonstrated above in the fluorescence and conductivity experiments. Additionally, the cryo-TEM data show intact liposomes with ν = 0.4 complexed with SPBs, an observation inconsistent with the conductivity results that contrastingly show a release of NaCl solution from complexed ν = 0.4 liposomes. This suggests formation of local defects (“pointlets”) in the membranes of the complexed ν = 0.4 liposomes, leading to a slow release of salt but not to disruption of the liposomes visible by cryo-TEM.

■

CONCLUSIONS We have found that, by changing the content of anionic groups in the liposomal membrane, the integrity of liposomes complexed with SPBs and the stability of SPB/liposome complexes can be easily controlled. As follows from Table 1, where the obtained results are summarized, liposomes with ν from 0.2 up to 0.3 retain their integrity when binding to SPBs, and the resulting complexes are stable in a physiological solution with [NaCl] = 0.15 M. Such complexes, that contain 15−20 liposomes per SPB, seem to be promising carriers for biologically active compounds. F

dx.doi.org/10.1021/la3024265 | Langmuir XXXX, XXX, XXX−XXX

Langmuir

Article

(17) Kahler, H.; Lloyd, B. J. Density of polystyrene latex by a centrifugal method. Science 1951, 114, 34. (18) Sybachin, A. V.; Efimova, A. A.; Litmanovich, E. A.; Menger, F. M.; Yaroslavov, A. A. Complexation of polycations to anionic liposomes: Composition and structure of the interfacial complexes. Langmuir 2007, 23, 10034. (19) Yaroslavov, A. A.; Melik-Nubarov, N. S.; Menger, F. M. Polymer-induced flip-flop in biomembranes. Acc. Chem. Res. 2006, 39, 702. (20) Yaroslavov, A. A.; Sitnikova, T. A.; Rakhnyanskaya, A. A.; Ermakov, Yu.A.; Burova, T. V.; Grinberg, V.Ya.; Menger, F. M. Contrasting behavior of zwitterionic and cationic polymer bound to anionic liposomes. Langmuir 2007, 23, 7539. (21) Yaroslavov, A. A.; Rakhnyanskaya, A. A.; Yaroslavova, E. G.; Efimova, A. A.; Menger, F. M. Polyelectrolyte-coated liposomes: stabilization of the interfacial complexes. Adv. Colloid Interface Sci. 2008, 142, 43. (22) Borisov, O. V.; Zhulina, E. B.; Birshtein, T. M. Diagram of states of grafted polyelectrolyte layer. Macromolecules 1994, 27, 4795. (23) Pincus, P. Colloid stabilization with grafted polyelectrolytes. Macromolecules 1991, 24, 2912. (24) Nakano, K.; Tozuka, Y.; Yamamoto, H.; Kawashima, Y.; Takeuchi, H. A novel method for measuring rigidity of submicron-size liposomes with atomic force microscopy. Int. J. Pharm. 2008, 355, 203. (25) Reviakine, I.; Brisson, A. Formation of supported phospholipid bilayers from unilamellar vesicles investigated by atomic force microscopy. Langmuir 2000, 16, 1806. (26) San Juan, A.; Letourneur, D.; Izumrudov, V. A. Quaternized poly(4-vinylpyridine)s as model gene delivery polycations: structurefunction study by modification of side chain hydrophobicity and degree of alkylation. Bioconjugate Chem. 2007, 18, 922. (27) Yaroslavov, A. A.; Efimova, A. A.; Sybachin, A. V. Effect of the phase state of the lipid bilayer on the structure and characteristics of the polycation-(anionic liposome) complex. Polym. Sci., Ser. A 2009, 51, 638.

G

dx.doi.org/10.1021/la3024265 | Langmuir XXXX, XXX, XXX−XXX