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Phase Structures of a Hydrated Anionic Phospholipid Composition Containing Cationic Dendrimers and Pegylated Lipids Ajay J. Khopade,*,†,‡ Dinesh B. Shenoy,§ Surekha A. Khopade,‡,§,| and Narendra K. Jain| Max Planck Institute of Colloids and Interfaces, Am Mu¨ hlenberg 1, D-14476 Golm, Germany, Dr. Harisingh Gour University, Sagar 470 003 (M. P.), India, and Bouve´ College of Health Sciences, School of Pharmacy, Northeastern University, Boston, Massachusetts 02115 Received February 6, 2004. In Final Form: July 15, 2004 The effect of 4th generation poly(amidoamine) dendrimer (4G PAMAM) present in an anionic phospholipid composition, consisting of hydrogenated soyphosphatidylcholine (HSPC), cholesterol (CH), dicetyl phosphate (DCP), and poly(ethylene glycol) (Mw ∼ 2000) derivatized phosphatidylethanolamine (PEG2000-PE), on the hydration and liquid crystalline structure formation was investigated. The optical and polarized light microscopies of the liposomal dispersion obtained from the hydrated lipid composition show two types of birefringent structures (mesophases): plastic, wormlike microstructures and conventional, over-elongated lamellae. Differential scanning calorimetry (DSC) shows an increase in the liquid crystalline phase transition (Tg) of the lipid composition from 60 to 94 °C with increasing 4G PAMAM concentrations from 0 to 0.011 mM, respectively. The Tg values of the two microstructures were 68 and 84 °C, respectively, indicating that the plastic microstructures were 4G PAMAM/DCP-complexes-rich (R mesophases) and the conventional and elongated lamellae were dendrimer-doped HSPC/CH-rich microstructures (β mesophases). Optical microscopy shows that the R mesophases convert into various other types of vesicular structures such as giant unilamellar vesicles and biliquid foams, upon heating above the phase transition temperature of the lipid composition (∼60-65 °C). The microstructure transformation is a result of an osmotic influx of water and the detergent action of PEG2000-PE present in the lipid composition. The transmission electron microscopy (TEM) images of the liposomal dispersion show particles embedding circular transparent domains that exactly correlate to the theoretical 4G PAMAM/DCP complex sizes, thus, providing evidence of 4G PAMAM interspersed within the two mesophases. Small-angle X-ray scattering (SAXS) measurements indicate that the R mesophases are a dendrimer-interlinked, symmetrically undulated lamellar phase and the β mesophases are dendrimer-doped, occasionally kinked lamellae. An increase in dendrimer concentration in the lipid composition was found to decrease interlamellar spacing. On the basis of optical microscopy, DSC, TEM, and SAXS data, a model of dendrimer-doped mesophase structure and lamellae fusion is proposed. This investigation provides new self-assembled materials for drug/gene delivery and supplements the understanding of mechanisms involved in various biological processes such as membrane fusion, transmembrane permeation, and endocytosis.
Introduction Dendrimers are the family of polyelectrolytes characterized by a precise molecular weight (size) and low polydispersity and have a core-shell structure. The core can host small organic molecules or ions while the charged periphery can provide binding sites to a wide range of oppositely charged molecules and ions.1 Supramolecular complexes of dendrimers with surfactants and the interaction of dendrimer with supramolecular surfactant assemblies such as anionic micelle,2 anionic liposome,3 and lamellar phase4 have been thoroughly studied. The * Corresponding author. Phone: +91 265 2341400. Fax: +91 265 2339103. † Max Planck Institute of Colloids and Interfaces. ‡ Present address: Sun Pharma Advanced Research Centre, Akota, Baroda-390 020, Gujarat, India. § Bouve ´ College of Health Sciences. | Dr. Harisingh Gour University. (1) (a) Tomalia, D. A.; Esfand, R. Chem. Ind. 1997, 416-420. (b) Zang, F.; Zimmerman, S. C. Chem. Rev. 1997, 1681-1712. (2) (a) Ottaviani, M. F.; Andechaga, P.; Turro, N. J.; Tomalia, D. A. J. Phys. Chem. B 1997, 101, 6057-6065. (b) Ottaviani, M. F.; Turro, N. J.; Jockusch, S.; Tomalia, D. A. Colloids Surf., A 1996, 115, 9-21. (3) (a) Ottaviani, M. F.; Favuzza, P.; Sacchi, B.; Turro, N. J.; Jockusch, S.; Tomalia, D. A. Langmuir 2002, 18, 2347-2357. (b) Sideratou, Z.; Foundis, J.; Tsiourvas, D.; Nezis, I. P.; Papadimas, G.; Paleos, C. M. A. Langmuir 2002, 18, 5036-5039. (c) Ottaviani, M. F.; Favuzza, P.; Bigazzi, M.; Turro, N. J.; Jockush, S.; Tomalia, D. A. Langmuir 2000, 16, 7368-7372.
motivation behind these studies was to produce novel materials for drug/gene delivery5 and heavy metal extraction6 and to understand various biological processes, for example, membrane fusion and transport.7 The use of dendrimers for gene delivery5 and transmembrane drug delivery8 and as an antibacterial biotherapeutic agent9 relies on its membrane disrupting properties similar to other cationic macromolecules.10 It is interesting not only to understand the affect of dendrimers on biological membranes using liposomes and other mesophases as models2-4 but also to develop phospholipid-dendrimer complex-based rational materials for drug/gene delivery. (4) Li, X.; Imae, T.; Leisner, D.; Lo´pez-Quintela, M. A. J. Phys. Chem. B 2002, 106, 12170-12177 (5) (a) Zinselmeyer, B. H.; Mackay, S. P.; Schatzlein, A. G.; Uchegbu, I. F. Pharm. Res. 2002, 19, 960-967. (b) Bielinska, A. U.; Chen, C. L.; Johnson, J.; Baker, J. R. Bioconjugate Chem. 1999, 10, 843-850. (c) Tomlinson, E.; Rolland, A. P. J. Controlled Release 1996, 39, 357-372. (6) (a) Diallo, M. S.; Balogh, L.; Shafagati, A.; Johnson, J. H.; Goddard, W. A.; Tomalia, D. A. Environ. Sci. Technol. 1999, 33, 820-824. (b) Rether, A.; Schuster, M. React. Funct. Polym. 2003, 57, 13-21. (7) (a)Yang, L.; Huang, H. W. Biophys. J. 2003, 84, 1808-1817. (b) Yang, L.; Huang, H. W. Science 2002, 297, 1817-1818. (8) (a) Chauhan, A. S.; Sridevi, S.; Chalasani, K. B.; Jain, A. K.; Jain, S. K.; Jain, N. K.; Diwan, P. V. J. Controlled Release 2003, 90, 335-343. (b) El-Sayed, M.; Ginski, M.; Rhodes, C. A.; Ghandehari, H. J. Bioact. Compat. Polym. 2003, 18, 7-22. (9) (a) Chen, C. Z. S.; Cooper, S. L. Biomaterials 2002, 23, 33593368. (b) Chen, C. Z. S.; Beck-Tan, N. C.; Dhurjati, P.; van Dyk, T. K.; LaRossa, R. A.; Cooper, S. L. Biomacromolecules 2000, 1, 473-480.
10.1021/la049682k CCC: $27.50 © 2004 American Chemical Society Published on Web 08/06/2004
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Recently, we have prepared liposomes from an anionic phospholipid composition containing 4th generation poly(amidoamine) dendrimers (4G PAMAM), which facilitated loading and retarded release of an anionic drug.11 Optical microscopy of this liposomal dispersion revealed different types of structures in addition to the typical liposomes. The properties of these structures were similar to those of “plastic membranes” that are made up of a cationic polyelectrolyte and soy lecithin in a stoichiometric ratio.12 This work presents a detailed investigation of the microstructures formed upon hydration of a similar anionic phospholipid composition containing 4G PAMAM using optical and transmission electron microscopy (TEM), differential scanning calorimetry (DSC), and small-angle X-ray scattering (SAXS) measurements. The other interesting microscopic phenomena such as lamellae fusion, formation of giant liposomes, multivesicular liposomes,13 and biliquid foams (BLFs)14 resulting from heating the microstructures above the phase transition temperature of the lipid composition are also investigated. Relying on these investigations, we propose a model for the dendrimer-doped, metastable mesophase structures and giant and multivesicular liposome formation phenomena. Unlike previous publications that investigated the effect of dendrimer on a preformed, defined supramolecular system2-4 (micelles, liposomes, or lamellae), this study investigates different self-assembled structures formed from the hydration of a supramolecular system-forming composition containing dendrimer. Materials and Methods Purified hydrogenated soyphosphatidylcholine (HSPC) and poly(ethylene glycol) (Mw ∼ 2000) derivative of phosphatidylethanolamine (PEG2000-PE) were purchased from Lipoid GmbH, Germany. Cholesterol (CH), dicetyl phosphate (DCP), and 4G PAMAM as a 10% (w/v) methanolic solution were purchased from Sigma-Aldrich, U.S.A. Solvents and buffers were obtained from Merck, Germany. All the chemicals were used without any further treatment or purification Liposomal formulations were prepared by the conventional thin film hydration method.15 Purified HSPC, CH, and DCP in a 1.5:1:1 molar ratio (equivalent to 1.8 mM of lipid considering an average molecular weight of the lipids used) and 0.01 mM PEG2000-PE was dissolved in chloroform in a round-bottomed flask attached to a rotary vacuum evaporator (Bu¨chi). The solvent was evaporated under a controlled vacuum to form a uniform thin film without bubbles. The lipid film was then hydrated with 12 mL of Millipore water at 60 ( 2 °C for 1 h in a thermostatically controlled water bath. The liposomes were intermittently sonicated on a bath sonicator, which was also maintained at 60 ( 2 °C. Similarly, thin films containing 0-0.011 mM 4G PAMAM (0.011 mM 4G PAMAM is in a 1:1 stoichiometric ratio with DCP) in the above lipid compositions were prepared by adding an equivalent quantity of methanolic 4G PAMAM (as supplied) in the lipid mixture during the film formation. The film was hydrated with Millipore water as described above to obtain the dispersion of a dendrimer-liposome supramolecular system. The liposomes were observed under the optical microscope (Zeiss Axioscope 40, (10) (a) Huang, W. M.; Han, X. J.; Wang, E. K. J. Electrochem. Soc. 2003, 150, E218-E221. (b) Fischer, D.; Li, Y. X.; Ahlemeyer, B.; Krieglstein, J.; Kissel, T. Biomaterials 2003, 24 (7), 1121-1131. (c) Wagner, E. J. Controlled Release 1998, 53, 155-158. (d) Rittner, K.; Benavente, A.; Bompard-Sorlet, A.; Heitz, F.; Divita, G.; Brasseur, R.; Jacobs, E. Mol. Ther. 2002, 5, 104-114. (11) Khopade, A. J.; Caruso, F.; Tripathi, P.; Nagaich, S.; Jain, N. K. Int. J. Pharm. 2002, 232, 157-162. (12) Antonietti, M.; Kaul, A.; Thu¨nemann, A. Langmuir 1995, 11, 2633. (13) Mantripragada, S. Prog. Lipid Res. 2002, 41, 392-406. (14) (a) Sebba, F. J. Colloid Interface Sci. 1972, 40, 468-474. (b) Omotosho, J. A.; Whateley, T. L.; Law, T. K.; Florence, A. T. J. Pharm. Pharmacol. 1986, 38, 865-880. (15) Bangham, A. D.; Standish, M. M.; Watkins, J. C. J. Mol. Biol. 1965, 13, 238-252.
Germany) equipped with a fluorescence lamp and cross polarizers. The formation of liquid crystalline phases and microstructures was detected with the polarizer and analyzer set at 90°. TEM images were obtained with a Zeiss EM 912 OMEGA instrument operating at an acceleration voltage of 120 kV. Sample grids were prepared by applying one droplet of the ∼100 times diluted liposomal dispersion sample onto a carbon-coated 400mesh copper grid and left to dry. No electron stains were used to avoid possible interactions with the dendrimer.3c DSC was performed on a Netzsch DSC 200. The samples were examined at a scanning rate of 10 K min-1 by applying two heating and one cooling cycle between 0 and 100 °C. The peak from the second heating cycle was noted. A typical sample size was 20 mg taken from the dried lipid film in an aluminum pan for measurements. DSC was also performed for the air-dried samples 1 and 2 described later under sample preparation. The type of liquid crystalline microstructures was further identified using small- angle X-ray scattering (SAXS). SAXS curves were recorded by using a Nonius rotating anode (P ) 4 kW, Cu KR) with pinhole collimation. An imaging plate was used as a detection system for the SAXS measurements. The sampleto-detector distance was set at 40 cm. The X-ray exposure time was fixed at 24 h for each sample of liquid crystals to reduce the noise level and to obtain a sufficiently high scattering intensity. The scattering vector range covered was 0.05-1.6 nm-1 [s ) 2/(λ) sin (θ), scattering angle 2θ, λ ) 0.154 nm]. Two-dimensional diffraction patterns were transformed into a one-dimensional radial average of the scattering intensity. The smearing effect was considerably low for the pinhole collimator. The experimental data were corrected for background scattering. Samples for SAXS were prepared by centrifuging the liposomal suspension at various speeds to separate the two types of structures (see later). Initially, the dispersion was centrifuged at 300g for 10 min. The sediment consisting of large aggregates (plastic, gel-like structures) was carefully taken into glass capillaries (sample 1). The supernatant was again centrifuged at 3000g for 10 min. The sediment obtained from this centrifugation step was discarded. The supernatant was further centrifuged at 10 000g for 10 min. The sediment was taken into glass tubes (sample 2). These samples were kept at 30 °C in a water bath controlled by a thermostat for 24 h until equilibrium was attained and the sample became more concentrated. The glass tubes were then sealed and taken for measurement. The samples with various dendrimer contents were prepared as sample 2.
Results and Discussion The liposomal dispersions prepared from the phospholipid compositions with and without 4G PAMAM differ in their physical appearance. The ability of cationic 4G PAMAM to electrostatically interact with the oppositely charged lipids (DCP) and induce a phase change16 makes the dendrimer-containing dispersion appear curdy. This dispersion (most likely 4G PAMAM/DCP complexes) further tends to aggregate upon sonication in a bath sonicator, which could be dispersed again by thorough shaking. Two types of microstructures are observed under the optical microscope: aggregated, complex morphological structures and a combination of spherical, elliptical, and elongated threadlike structures. The aggregated structure tends to fuse, elongate, and break when sheared (mechanical stress) with a coverslip on a glass slide using a finger. Figure 1a shows sheared structures that look like worms revealing patterned birefringence (Figure 1b) under the cross-polarized light. They are much harder than the bare surfactant structures (without dendrimer) obtained from the corresponding lipid mixture, indicating that they possess reasonable material properties. The second type (16) (a) Paulo, S. K.; Yan, L.; Marcia, C. B. Chem. Phys. Lett. 1998, 298, 51-56. (b) Iolanda, P.; Rosa, G.; Clara, G.; Agustı´n, C.; Concepcio´n, A. Polymer 1997, 38, 5107-5113.
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Figure 1. Optical microscopy images of various structures formed after the hydration of 4G PAMAM containing an anionic lipid composition: (a) wormlike structures formed by shearing aggregates (4G PAMAM/DCP complexes) in the hydrated lipid dispersion, (b) patterned birefringence presented by wormlike structures indicating optical anisotropy, (c) elongated lamellar threads with a nonuniform aqueous phase distribution between the bilayers, and (d) birefringent Maltese crosses and streaks confirming the lamellar phase of the threadlike structures. Multiple kinks are seen in the birefringent thread. At terminals, the thread tends to bend into the closed vesicular structures. The Tg value of the structures in parts a and b was 84 °C and that for parts c and d was 68 °C.
of structures, shown in Figure 1c,d, are typical lamellar phases consisting of bilayers of phospholipid confirmed by the typical Maltese crosses and streaks under the polarized light. The lamellae tend to elongate and bend into small, spherical, birefringent particles or partially fused structures with/without any shear application, which resembled conventional liposomes. The dendrimer molecules present in the aqueous domain at a concentration less than the critical dendrimer concentration4 produced kinks in the elongated lamella (see later). The relative abundance of the two structures depends on the amount of 4G PAMAM present in the anionic lipid composition: the liposomal structures are found in abundance when the amount of 4G PAMAM in the phospholipid composition is less and the curdy dispersion appears on increasing the 4G PAMAM concentrations. This qualitatively suggests that the interaction of 4G PAMAM and DCP to form 4G PAMAM/DCP complexes induces microstructure and phase changes in the lamellae.16 The DSC thermograms from the phospholipid compositions with an increasing amount of 4G PAMAM from 0 to 0.011 mM show an almost linear increase in the phase transition temperature, Tg, from 60 to 94 °C. This was a liquid crystalline phase transition, because a broad enthalpic phase transition was obtained for each of the dendrimer-containing lipid compositions, and the birefringence is also preserved above this temperature. An increase in the Tg value was a clear indication of the 4G PAMAM/DCP complex formation in the lamellar mesophases and correlates well with the previous DSC measurements on dipalmitoylphosphatidylglycerol/ cationic polyelectrolyte complexes of varying stoichiometric ratios.17 The Tg of the two structures (wormlike and lamellar) formed with the present liposomal composition were 84 and 68 °C, respectively. The PAMAM/DCP complexes in a 1:1 stoichiometric ratio without any neutral lipids in the composition did not show an endothermic peak in the range studied (0-100 °C). The DSC data presented above qualitatively suggest that the two microstructures consist of the 4G PAMAM/DCP-complex(17) Tirrell, D. A.; Turek, A. B.; Wilkinson, D. A.; McIntosh, T. J. Macromolecules 1985, 18, 1512.
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Figure 2. Optical microscopy images of the BLF structure and GUV formation on heating the stage to 65 °C. The Tg of the lipid composition used for making this liposomal dispersion was 84 °C. The images 1-4 were captured over a period of 30 min at 10-min intervals. Image 5 was captured after another 45 min. Image 6 shows multiple GUVs budding off from the R mesophase particles.
rich (R) and HSPC/CH-rich (β) domains.18 In the wormlike structure (designated R mesophases), R > β, while in the lamellar structure (designated β mesophases), β > R. The anisotropic distribution of lipids (R > β domains) within microstructures leads to the formation of various other metastable morphological structures on heating the liposomal dispersion above the phase transition temperature of the lipid mixture.18a Figure 2 (images 1-5) shows the metastable structure formation process under the optical microscope. The microstructure tends to swell and fuse within 10 min and, in another 20 min, slowly grows into the multivesicular vesicles or BLF structures (Figure 2, image 5).13,14 The fusion process continues over a long period, ultimately forming a giant foamlike and unilamellar vesicular structure. Unifocal lamellar fusion and swelling in the smaller particles form giant unilamellar vesicles (GUVs; Figure 2, image 6). The pure 4G PAMAM/ DCP complexes in a 1:1 stoichiometric ratio (Tg > 100 °C) do not form the giant or multivesicular vesicles. This observation supports the presence of R and β domains within the two structures. An increase in the rate of vesicle budding and fusion was observed above the liquid crystalline phase transition temperature of the pure lipid composition (∼65 °C) on a hot-stage microscope. From the microscopic observations, it was concluded that the formation of GUVs and BLFs initiate with the temperature-induced melting of the 4G PAMAM interlinked lamellar phase (see later) facilitated by an osmotically driven influx of water into the vesicles and the detergent action of the PEG2000-PE on the bilayers. The 4G PAMAM and polar headgroups of the lipids attract water by osmosis,19 and a high HLB value of PEG2000-PE imparts the detergent action.20 The absence of PEG2000-PE in the composition delays the formation of such distinct structures. Osmotic swelling induces random fusion of (18) (a) Lipowsky, R.; Dimova, R. J. Phys.: Condens. Matter 2003, 15, S31-S45. (b) Antonietti, M.; Wenzel, A.; Thunemann, A. Langmuir 1996, 12, 2111-2114. (19) Cationic polymers, including PAMAM, are potential endosomolytic polymers; that is, once they are internalized in endosomes, a lot of water is pumped inside them and they are lysed. (a) Cho, Y. W.; Kim, J. D.; Park, K. J. Pharm. Pharmacol. 2003, 55, 721-734. (b) Griffiths, P. C.; Paul, A.; Khayat, Z.; Wan, K. W.; King, S. M.; Grillo, I.; Schweins, R.; Ferruti, P.; Franchini, J.; Duncan, R. Biomacromolecules 2004, 5, 1422-1427. (20) Belsito, S.; Bartucci, R.; Sportelli, L. Biophys. Chem. 2001, 93, 11-22.
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Figure 3. (a) TEM image of a liposomal dispersion prepared from an anionic phospholipid composition containing 4G PAMAM. A drop of the dispersion was dried on a copper grid for TEM observations. (b) Transparent circles correspond to the size of the 4G PAMAM/DCP complexes embedded within the R mesophase particles; (c) a R mesophase particle with >60% of the area occupied by dendrimer/DCP complexes.
the dendrimer interlinked lamellar structures inside the vesicles through a “stalk”7 pathway to form BLF structures. The swelling rate of BLFs slows down significantly on adding sodium chloride (0.15 M) in the external medium. In contrast to the earlier investigations,7,21 which do not support the role of osmotic pressure in dendrimermediated fusion of membranes, we show that osmotic swelling has indeed an important role in membrane fusion of the bilayer foam cells. This mechanism partly explains the reason for the high encapsulation and slow release of an anionic drug obtained in our previous studies from a similar liposomal composition.11 To investigate the microstructural details of the two types of mesophases explained above, TEM and SAXS measurements were performed. The sizes of two types of structures ranged from a few hundred micrometers to a few hundred nanometers, as measured from optical microscopy (Figures 1 and 2) and TEM (Figure 3), respectively. TEM images in Figure 3a,c show tiny, round, transparent domains embedded within nanometer-sized elongated and spherical structures, respectively. Considering 4.5 nm as the 4G PAMAM diameter (www.dendritech.com) and ∼2.5 nm for the interacted phospholipid monolayer12-22 surrounding the dendrimer, a theoretical diameter of ∼9.5 nm is obtained for 4G (21) (a) Zhang, Z. Y.; Bradley, D. S. Bioconjugate Chem. 2000, 11, 805-814. (b) Karoonuthaisiri, N.; Titiyevskiy, K.; Thomas, J. L. Colloids Surf., B 2003, 27, 365-375. (22) Charitat, T.; Bellet-Almalric, E.; Fragneto, G.; Graner, F. Eur. Phys. J. B 1999, 8, 583.
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PAMAM/DCP complexes, which correlates with the size of round transparent domains in the TEM images (Figure 3a,b). The dendrimer distribution within R mesophases contributes to more than 60% of the area (Figure 3c) while the scattered dendrimers (
