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Novel Cationic Amphiphilic Derivatives from Vernonia Oil: Synthesis and Self-Aggregation into Bilayer Vesicles, Nanoparticles, and DNA Complexants S. Grinberg,*,† C. Linder,† V. Kolot,† T. Waner,‡ Z. Wiesman,† E. Shaubi,† and E. Heldman‡ The Institutes for Applied Research, and The Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel Received January 12, 2005. In Final Form: May 19, 2005 Self-assembling nanostructures were prepared from novel cationic amphiphilic compounds synthesized from vernonia oil, a natural epoxydized triglyceride. The presence of a 12,13-epoxy group on the C18 unsaturated fatty acid, vernolic acid, which is the main constituent of vernonia oil, permitted the synthesis of novel amphiphilic derivatives with a hydrogen-bonding hydroxyl and a cationic headgroup moiety on adjacent carbon atoms. The amphiphiles were prepared in a two-stage synthesis that comprised opening of the epoxy groups with a haloacetic acid, followed by quaternization of the halo group with a tertiary amine containing a C12 aliphatic chain. Intact vernonia oil as the starting material gave a triple-headed cationic amphiphile, containing three vernolic acid derived moieties connected through a glycerol backbone. A single-headed amphiphile with two alkyl chains and a single quaternary ammonium headgroup was synthesized from the methyl ester of vernolic acid as the starting material. The triple-headed derivative could form nonencapsulating structures. Cholesterol was required in the formulation (1:1) to make spherical vesicles that could encapsulate a water-soluble marker. The single-headed derivative, however, formed spherical encapsulating vesicles without cholesterol. TEM, NMR, and FT-IR were used to characterize the vesicles, and molecular structure vs morphology relationships were postulated on the basis of these data. The triple-headed amphiphile also formed a DNA complex that was highly resistant to hydrolysis by DNase. This amphiphile-DNA complex was used as vector for gene transfer in cell culture demonstrating efficient DNA transfection.
Introduction Phospholipids characterized by two long aliphatic chains, an ionic or amphoteric headgroup, and an interface region (glycerol) connecting these two moieties are wellknown amphiphilic compounds that form vesicles.1,2 Other classes of vesicle-forming amphiphiles are ammonium salts having two long alkyl chains but no chemically distinctive interface region3 and many other compounds,4-8 including single-tail amphiphiles with rigid segments and flexible tails9 and amphiphiles with three alkyl chains and two headgroups.10,11 Empirical data indicate that the morphology of the aggregated structures (micelles, liposome-type vesicles or inverse micelles) is controlled by a balance between the * To whom correspondence should be addressed. E-mail: sarina@ bgumail.bgu.ac.il. † The Institutes for Applied Research. ‡ The Faculty of Health Sciences. (1) Fendler, J. H. Membrane Mimetic Chemistry; John Wiley & Sons: New York, 1982; Chapter 6 “Vesicles”. (2) New, R. R. C. Liposomes: A Practical Approach; Oxford University Press: New York, 1993. (3) Kunitake, T.; Okahata, Y. J. J. Am. Chem. Soc. 1977, 99, 3860. (4) Kunitake, T.; Okahata, Y. J.; Shimomura, M.; Yasunami, S.; Takarabe, K. J. Am. Chem. Soc. 1981, 103, 5401. (5) Fuhrhop, J-H.; Mathieu J. Angew. Chem., Int. Ed. Engl. 1984, 23, 100. (6) Kunitake, T. Angew. Chem., Int. Ed. Engl. 1992, 34, 709. (7) Menger, F. M.; Gabrielson, K. D. Angew. Chem., Int. Ed. Engl. 1995, 34, 2091. (8) Kunitake, T.; Nakashima, N.; Shimomura, M.; Okahata, Y.; Kano, T.; Ogawa, T. J. Am. Chem. Soc. 1980, 102, 6642. (9) Kaler, E. W.; Murthy, A. K.; Rodriguez, B. E.; Zasadzinski, J. A. Science 1989, 245, 1371. (10) Sumida, Y.; Masuyama, A.; Takasu, M.; Kida, T.; Nakasutji, Y.; Ikeda, I.; Nojima, M. Langmuir 2000, 16, 8005. (11) Sumida, Y.; Masuyama, A.; Takasu, M.; Kida, T.; Nakasutji, Y.; Ikeda, I.; Nojima, M. Langmuir 2001, 17, 609.
attractive forces of the aliphatic chains, the repulsion forces of the headgroups, and the geometric molecular packing parameters of the amphiphiles.12 The relationship between amphiphile molecular parameters and vesicle characteristics (e.g.,vesicle size, shape, and morphology) can serve as a guide to the synthesis of new derivatives.13,14 A promising potential application of cationic liposomes is their use in gene therapy for transfecting cells with genetic material. These liposomes appear to be superior to virial vectors, since they are not infective and do not induce an immune response. It appears that cationic liposomes and vesicles interact with the negatively charged phosphate group(s) of the nucleotides. The balance between the molecular parameters, such as cationic charge density, position and length of aliphatic chains, and the presence of other groups is known to affect the efficiency of gene transfection.15,16 For the preparation of stable vesicles and DNA complexants, our group is currently investigating novel amphiphiles, having both hydrogen-bonding hydroxyl and cationic headgroups, that are synthesized from vernonia oil, a multifunctional natural oil. This oil is obtained from the seeds of Vernonia galamensis, a shrub of the Compositae family, native to northern and central Africa. The oil contains about 40% (of dry weight) of the triglyceride vernonia oil (Scheme 1), which is rich in an epoxy fatty (12) Israelachvili, J. Intermolecular and Surface Forces, Second Edition, Academic Press: New York, 1992; Chapter 17. (13) Winterhalter, M.; Lasic, D. D. Chem. Phys. Lipids 1993, 64, 35. (14) Lasic, D. D. Stealth Liposomes. In Microencapsulation Methods and Industrial Applications; Benita, S., Ed.; Dekker: New York, 1996; Chapter 11, p 302. (15) Simberg, D.; Hirsch-Lerner, D.; Nissim, R.; Barenholtz, Y. J. Liposome Res. 2000, 10, 1. (16) Xiaohuai; Huang Biochim. Biophys. Acta 1994, 1189, 195.
10.1021/la050091j CCC: $30.25 © 2005 American Chemical Society Published on Web 07/14/2005
Derivatives from Vernonia Oil Scheme 1a
a
Trivernolin is the main triglyceride of vernonia oil
acid, commonly known as vernolic acid II (cis-12,13-epoxy, cis-9-octadecenoic acid).17,18 The synthesis of two amphiphilic derivatives based on vernonia oil and their vesicle forming and DNA transfection characteristics is described in this paper. Structurally, both derivatives have a vernolic acid moiety with quaternary ammonium headgroups. The first derivative, VI is synthesized from methyl vernolate III and contains only one vernonia moiety. The second derivative, VII is synthesized from vernonia oil I and contains three vernolic acids moieties bound together through a glycerol backbone.
Experimental Section Materials. Vernonia oil (70% of vernolic acid, as determined by gas chromatography; 2.1 epoxy functionalities per molecule) was obtained from Ver-Tech, Inc. (Bethesda, USA). Chemical reagents and solvents were purchased from Aldrich Chemical Co. Epoxy groups were determined by potentiometric titration.19 FT-IR analysis was carried out on a Nicolet spectrometer. 1Hand 13C NMR spectra were determined with Brucker WP-200 SY and Brucker WP-500 SY spectrometers, respectively, in CDCl3 solution with TMS as the internal standard. HPLC analysis was carried out on a C18RP column with an evaporative light scattering detector (evaporation temperature 46 °C; mobile phase methanol:water 9:1 (v/v); flow rate 0.5 mL/min). Vesicle Preparation. Vesicles were prepared from vernonia oil derivatives by the method generally used to prepare liposomes or vesicles, i.e., ethanol injection or film hydration followed by bath sonication.2 The size and shape of the vesicles were dependent on the headgroup counterion used (Cl- or Br-) and (17) Gundstone, F. D. J. Chem. Soc. 1954, 1611. (18) Carlson, K. D.; Schneider, W. J.; Chang, S. P.; Princen, L. H. In New Sources of Fats and Oils; Pryde, E. H., Princen, L. H., Mukherjee, Eds.; American Oil Chemists Society: Champain, IL, 1981; pp 297318. (19) Jay, R. R. Anal. Chem. 1964, 36, 667.
Langmuir, Vol. 21, No. 17, 2005 7639 the composition of the medium (either deionized water, 0.1 M NaCl, or Tris-buffer at pH 7.4 containing 50 mM NaCl and 5 mM histidine). In a typical ethanol injection procedure, 1 mL of ethanolic solution of the amphiphile (5 mM) was slowly added to 24 mL of deionized water (DI) or Tris-buffer at room temperature. Then the mixture was sonicated in a bath at 25 °C for 30 min to form vesicle dispersion. In some experiments, 2% uranyl acetate was added to the solution as a marker for encapsulation. Since the derivatives contain unsaturated aliphatic chains, which exhibit a gel to liquid crystal transition at temperatures below 0 °C, all procedures for vesicle formation were carried out at 25 °C. Characterization of the Vesicles Transmission Electron Microscopy (TEM). TEM was conducted on a JEOL-100CX instrument. A drop of the vesicle suspension was placed on a Formvar-coated copper grid, airdried, negatively stained with a drop of 0.5% uranyl acetate solution in water, and dried again at room temperature. Dynamic Light Scattering (DLS). All samples were prefiltered through a 200-micron microfiltration membrane. The solution sample was injected into a thin-walled cylindrical borosilicate glass cuvette (1 cm diameter) and placed in a vat filled with analytical grade pure toluene as the index matching fluid. During the course of the measurements, the vat temperature was kept at ∼20 °C. The light source was an argon ion laser (Spectra Physics-Lexel, λ ) 514.5 nm), and photons scattered by the sample were collected by a photomultiplier tube mounted on the goniometer arm at 90° to the direction of the incident radiation. The photoelectron count-time autocorrelation function was measured with a BI 2030AT (Brookhaven Instruments, Holtsville, NY) digital correlator and analyzed using a cumulant expansion and/or the constrained regularization algorithm, CONTIN. The results are given as an intensity-weighted distribution of effective diffusion coefficients. An effective hydrodynamic radius is calculated from the mean diffusion coefficient by using the Stokes-Einstein relationship. FTIR. The vesicle samples were spread on CaF2 and their spectra was determined with a Nicolet spectrometer instrument. NMR. 1H NMR spectra were determined with a Brucker WP200 SY spectrometer. Static Turbidimetry. A 2.5-mL sample of vesicle suspension in 4% ethanolic water was placed in a quartz cuvette. The turbidimetric profile was recorded in the range of 300-600 nm with a UV-vis Hewlett-Packard HP-8452A spectrophotometer at 25 °C. Ethanolic water (4%) was used as reference. Gene Transfection. For complexation and encapsulation of DNA,20 vesicles were prepared as follows. A thin film of a vernonia oil derivative was prepared by dissolving 5 mg of the derivative in 5 mL of chloroform:methanol (1:1, v/v) in a 50-mL roundbottom flask. The solvent was removed under reduced pressure with a rotavapor, and the thin film so obtained was dried overnight in a vacuum desiccator to remove traces of solvent. Then 1 mL of phosphate-buffered saline containing 0.1 mg of the EGFP-N1 reporter gene encoding a red-shift variant of the wildtype green fluorescent protein (GFP) was added to the flask and the content was stirred with a vortex mixer to form vesicles containing the reporter gene. COS-7 cells, used as target cells, were grown in 96-well plates or in 30-mm Petri dishes to 4050% confluency. Transfection with DEAE-dextran was used both as a positive control and as a reference method. Transfection efficiency was determined by counting the number of transfected cells (green fluorescent cells) per total number of cells seen in the same field by a fluorescent microscope.
Results and Discussion Synthesis. The synthetic strategy for the formation of amphiphilic derivatives based on vernonia oil was the use of the C12-C13 epoxy group of vernolic acid for the introduction of a quaternary ammonium headgroup and a hydroxyl group. The amphiphilic derivatives were prepared in a two-step synthesis. The first step comprised a nucleophilic attack of a monohalogen carboxylic acid on (20) Washbourne, P.; McAllister, A. K. Curr. Opin. Neurobiol. 2002, 12 (5), 566.
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Scheme 2
Scheme 3
Table 1. Chemical Shifts for IVa in the 1H and Spectra
group 1. CH-OH 2. CH-O-C(O) 3. CH-O-C(O) 4. CH2-Cl
δ ppm 1H NMR 3.58 4.8-4.9 4.04-4.09
13C
NMR
Scheme 4
δ ppm 13C NMR 71.87; 71.98 78.29 167.09; 167.24 41.04
the epoxy groups with the formation of a hydroxy ester as the main product. In the second step, the haloacetate derivative was quaternized with the tertiary amine DMDA that contains a long aliphatic chain. When this procedure was applied to the methyl ester of vernolic acid III, a single-headed double-chain amphiphile VI was formed. When the reaction was carried out on vernonia oil I (Scheme 1), the final derivative VII contained three of the single headed amphiphiles bound to a glycerol backbone. Synthesis of the Haloacetate Derivative of Methyl Vernolate. Methyl vernolate III was allowed to react with a small excess of a halo (chloro- or bromo) acetic acid (Scheme 2). The chloro- and bromoacetate derivatives of methyl vernolate, IV (a and b, respectively), were isolated from the reaction mixture by column chromatography using a mixture of n-hexane and diethyl ether as the eluent with 70% yield. The IR spectra of the products showed the disappearance of the absorption bands at 820 and 840 cm-1 characteristic of the epoxy group and the appearance of new peaks characteristic of the hydroxyl group at 3450 cm-1 and of the haloacetate group at 1280 cm-1. The new signals in the NMR spectra of IVa are presented in Table 1. It is noteworthy that in the 1H NMR spectrum, the methylene protons of the chloroacetate group (4.04-4.09 ppm) appear as a multiplet and not as a singlet (4.03 ppm) observed in the corresponding derivative of castor oil,21 probably due to the formation of positional isomers. The formation of such isomers could also be seen in the 13 C NMR spectrum. The carbon atom adjacent to the hydroxylic group appears as two peaks, at 71.87 and 71.98 ppm. Two peaks, at 167.09 and 167.24 ppm, were also found for the carbonyl carbon of the new ester group in the chloroacetate derivative IVa. The double bond gave rise to four peaks at 123.03, 124.01, 133.71, and 133.96 ppm, thus confirming the formation of positional isomers. The 1H and 13C NMR spectra of the bromoacetate of methyl vernolate, IVb, are analogous to those of the chloroacetate derivative IVa described above. However, the signals of both the protons and the carbon atom of the methylene group adjacent to the bromine atom were (21) Baydar, A.; Rohnston, K. K. Int. J. Cosmet. Sci. 1991, 13, 169.
Scheme 5
shifted to a higher magnetic field, 3.85 and 25.26 ppm, respectively. Synthesis of the Haloacetate Derivatives of Vernonia Oil. The corresponding haloacetates of vernonia oil, V (a and b), were prepared in a similar manner as the haloacetates of methyl vernolate (Scheme 3). Vernonia oil was reacted with an excess of a haloacetic acid in dry toluene at 90 °C for 22-24 h to give a product yield of 85%. The course of the reactions was monitored by determination of the epoxy groups in the reaction mixture. The IR spectra of the chloroacetate derivative, Va, and of the bromoacetate derivative, Vb, of vernonia oil showed exactly the same new characteristic bands (3450, 1300, and 1280 cm-1) as the chloro- and bromoacetate of methyl vernolate (IVa and IVb, respectively). Likewise, the 1H NMR and 13C NMR spectra of the chloroand bromoacetate of vernonia oil exhibited the same new signals that were observed in the NMR spectra of the chloro- and bromoacetate of methyl vernolate. In these derivatives too, positional isomers were apparently formed, as indicated by the multiplet of the methylene protons of the haloacetate group seen in the 1H NMR spectra and the two peaks of the carbon atom adjacent to the OH group (CH-OH) seen in the 13C NMR spectra. Quaternization of the Haloacetate Derivatives of Methyl Vernolate and of Vernonia Oil. The haloacetates of methyl vernolate and of vernonia oil were quaternized with stoichiometric amounts of DMDA to give
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Table 2. Chemical Shifts for Ia in the 1H and
group 1. CH3-O 2. CH3-O-C(O) 3. CHdCH 4. CH-OH 5. CH-O-CO-CH2-N+(CH3)2 6. O-C(O)-CH2-N+(CH3)2 7. CO-CH2-N+(CH3)2 8. N+(CH3)2 9. N+(CH3)2-CH2-CH2
δ ppm 1H NMR 3.60 5.44-5.92 3.57 4.88-4.96 4.55-4.71; 5.44-5.52 3.48-3.50 3.63-3.73
the amphiphilic derivative VI, based on the methyl vernolate (Scheme 4), and derivative VII, based on vernonia oil (Scheme 5). The reactions were carried out in acetone or 2-propanol. The chloroacetates of methyl vernolate and of vernonia oil were less reactive than their corresponding bromoacetate derivatives. The quaternary ammonium salts based on the methyl vernolate (VIa and VIb) were isolated from the reaction mixture by column chromatography in a 30-60% yield with a purity of more than 99%. The quaternization degree for the quaternary ammonium salts based on vernonia oil VIIa and VIIb was in the range of 96-99.5%, as determined by argentometric titration. The structure of these quaternary ammonium compounds was determined by IR, NMR, and elemental analysis. IR spectra of the quaternary ammonium compounds VI and VII, showed that the introduction of the quaternary ammonium group resulted in the disappearance of the absorption peak at 1280 cm-1, characteristic of the haloacetate group, and the appearance of new peaks at 3350, 1235, and 1200 cm-1. The chemical shifts of the main groups in the 1H NMR and 13C NMR spectra of the
13C
NMR Spectra
δ ppm 13C NMR 51.99 174.23 123.46; 124.67; 132.06; 133.19 71.34; 71.68 79.76; 79.84 164.71; 164.89 61.52 51.39; 51.55 64.66
amphiphilic derivatives based on methyl vernolate VIa, (X ) Cl-) are presented in Table 2. 1 H and 13C NMR spectra showed the disappearance of the signals at 4.04-4.09 and 41.04 ppm characteristic of the methylene protons and carbon atom of the chloroacetate group and the appearance of new signals characteristic of the quaternary salt (Table 1). CH-COSY NMR spectrum of the quaternary derivative of methyl vernolate with Cl- anion VIa is presented in Figure 1. It is noteworthy that the carbon atom (Table 2 run 7) of the methylene group between the quaternary nitrogen and the carbonyl group O-CO-CH2-N+(CH3)2 may be correlated to two different chemical shifts in the proton NMR spectrum, at 4.55-4.71 ppm (H-7) and at 5.44-5.42 ppm, adjacent to the double-bond protons. The NMR spectra of the amphiphiles VI and VII clearly show that the positional isomers, characteristic of the chloroand bromoacetates of methyl vernolate (IVa and IVb) and of vernonia oil (Va and Vb), are retained in the quaternary ammonium compounds as well. The NMR spectra of the quaternary ammonium compounds prepared from the bromoacetate of methyl vernolate and chloro-
Figure 1. CH-COSY NMR spectrum of the quaternary derivative of methyl vernolate with Cl- as the counterion (VIa).
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Figure 2. TEM micrographs of vesicles prepared from VIb by film hydration and sonication method in a solution without and with uranyl acetate: (a) empty vesicles; (b) vesicles encapsulating uranyl acetate.
Figure 4. TEM micrographs of vesicles prepared by the method of ethanol injection in water from VIIa (a), VIIb (b), and by ethanol injection in Tris buffer at pH 7.4 from IIa (c).
Figure 3. Turbidity measured by absorption at 400 nm of vesicles prepared in DI water from VIb as a function of time elapsed after vesicle preparation.
and bromoacetates of vernonia oil were shown to have analogous structures. Formation and Characterization of the Vesicles Transmission Electron Microscopy and Dynamic Light Scattering of the Vesicles. The quaternary methyl ester derivative VIb formed vesicles, 50-200 nm in diameter, by film hydration and sonication in DI water (Figure 2a). In the presence of 2% uranyl acetate, vesicles of about 200 nm encapsulating the marker were formed (Figure 2b). There was a small change in the turbidity of the vesicle solutions in DI water up to 8 days which then remained relatively constant over a period of 28 days (Figure 3). Hence, we concluded that the dispersion was relatively stable and that only a relatively small amount of vesicle fusion took place. Derivative VII with three cationic headgroups and six long aliphatic chains is unique with respect to the number of headgroups and aliphatic chains. The conventional steric requirements for forming encapsulating vesicles may not be met by the molecular structure of this derivative. Using various formation methods, we investigated if this derivative forms nanovesicles or nanoparticles. Dispersion of this derivative by the ethanol injection method into DI water was seen in the TEM as a population of nanoparticles of approximately 20-80 nm in diameter (Figure 4a), when Cl- was used as the counterion (VIIa), and as a homogeneous population ranging from 30 to 40 nm in diameter (Figure 4b), when Br- was used as the counterion (VIIb). When tris-saline buffer was used instead of DI water, a very heterogeneous vesicle population of 30 to 400 nm was produced (Figure 4c). DLS measurements of the vesicles formed from VIIb by film hydration and sonication in DI water showed initially a vesicle population in the size range of 40-120
Figure 5. Size distribution of vesicles obtained by film hydration-sonication from VIIb measured by DLS.
nm, with a mean of 78 nm (Figure 5). The mean size of 71-76 nm was maintained over a period of 28 days, indicating that no vesicle aggregation occurred during that time. Numerous attempts to encapsulate a marker with the triple-headed cationic derivative (VIIa) by ethanol injection into a 2% uranyl acetate solution in DI proved unsuccessful. In TEM micrographs, no vesicles were seen to contain a black uranyl acetate core, which would have indicated encapsulation. Encapsulation also failed when the film hydration-sonication method was used with a 2% uranyl acetate solution. TEM micrographs showed a heterogeneous population of small to large particles 20300 nm in diameter (Figure 6a). The large particles had a multilamellar structure (Figure 6b,c) comprising about 20-nm-thick lamellae. The smaller particles seemed to have been formed by budding from the larger particles, as seen in Figure 6a,b. Derivative VIIa formed, however, spherical vesicles 50 and 200 nm in diameter that were capable of encapsulating uranyl acetate upon the addition of cholesterol to the amphiphile formulation in a 1:1 molar ratio (Figure 7). FT-IR and NMR. Since amphiphile VII is made from three vernolic acid derivatives bound together through a glycerol backbone and amphiphile VI from one, similarities in the FT-IR and NMR are expected and dissimilarities should help to explain differences in inter- and intramolecular forces. The strong absorption bands at 2925 and 2854 cm-1 for both amphiphiles may be assigned to
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Figure 6. TEM micrographs of vesicles prepared from VIIa by film hydration with a 2% solution of uranyl acetate and sonication at different sites and magnifications.
Figure 7. TEM micrograph of vesicles prepared from VIIa mixed with cholesterol in a 1:1 molar ratio by film hydration with a 2% solution of uranyl acetate.
the antisymmetric and symmetric CH2 stretching modes of all-trans chains22-24 and indicate that the methylene chains are subject to some short-range disorder, similar to that existing in the liquid crystalline state. The ratio between the two strong peaks, which was almost identical for the two amphiphilic derivatives, seems to be related to the intermolecular chain-packing order. In vesicles there was, however, a slight difference in hydrogen bonding between derivative VI and VII: the O-H stretching of the latter was shifted to a lower frequency at 3340 cm-1, indicating a stronger hydrogen bonding in derivative VII. FT-IR measurements of the derivative in CCl4 as a function of the concentration showed a shift in the O-H stretching peak to lower frequencies at higher concentrations. Hydrogen bonding in this molecule can be expected to be both intra- and intermolecular. The shift to lower frequencies may be attributed to intramolecular hydrogen bonding, whereas at higher concentrations, both intra- and intermolecular hydrogen bonding would result in even a lower shift of frequencies. In NMR spectra, restricted molecular motion is seen in amphiphilic molecule aggregates, as compared to solution. 1H NMR line broadening indicates tighter molecular packing within the membrane, causing broadening of the line width as compared to solution.4,22,25 Restriction of molecular motion were observed in the 1H NMR spectrum of the quaternary methyl vernolate VI, in D2O was compared with its spectrum in CDCl3 solution (Figure 8). In CDCl3, sharp peaks of the different protons are in contrast to the broader peaks seen in the D2O spectrum, (22) Wang, X.; Shen, Y.; Pan, Y.; Liang, Y. Langmuir 2001, 17, 3162. (23) Lu, X.; Zhang, Z.; Liang, Y. Langmuir 1997, 13, 533. (24) Bhattacharya, S.; Acharya, S. N. G. Chem. Mater. 1999, 11, 3121. (25) Zhang, Z.; Wu, L.; Liang, Y.; Yin, Q. J. Colloid. Interface Sci. 1997, 188, 501.
Figure 8. CDCl3.
1H
NMR spectra of vesicles from VIb in D2O and
Figure 9. 1H NMR spectra of vesicles prepared from the quaternary methyl vernolate amphiphile VIb (a) and the quaternary vernonia oil VIIb (b) in D2O solution at different temperatures.
suggesting aggregation into vesicles and hence restricted molecular motion in the latter. A further difference between derivatives VI and VII is seen in the stronger and sharper NMR peak for the N-methyl group (∼3.5 ppm) in derivative VI (Figure 9a at 30 °C) as compared with the peak of the same group in derivative VII (Figure 9b at 30 °C), which suggests that the headgroup of the former has greater mobility in water than that of the latter. In contrast, the narrow NMR signals of the methylene groups are alike for the two compounds, indicating that the alkyl chains of both
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derivatives are in the form of aggregates having similar chain mobility, as was also confirmed by the FT-IR spectra. 1 H NMR spectra of the vesicles revealed changes in the width of the proton absorption peak as the temperature increased from 30 to 75 °C; that is, the NMR peaks become narrower and stronger at elevated temperatures (Figure 9a,b). At 30 °C, the signal of the three N-methyl headgroups of derivative VII is an almost indiscernible very small broad signal. This broad signal becomes narrower as the temperature increased to 50 °C, due to higher headgroup mobility. A similar temperature behavior of the single N-methyl headgroup of derivative VI was observed with the signals, however being sharper at all of the temperatures. No other changes were observed when the temperature was increased to 75 °C. Increasing the temperature also causes a downfield shift of the NMR signals, probably as a result of the increasing interactions of the amphiphilic derivatives with water molecules.22 Amphiphile Structure-Vesicle Morphology Relationships. Derivative VII is composed of essentially three of the same moieties derived from vernolic acid and bound together through a glycerol backbone, whereas derivative VI contains only one such moiety. The vernonia moiety is a C18 aliphatic chain with a C9-C10 double bond, a C12 or C13 hydroxyl group, and a C12 or C13 quaternary ammonium salt as the headgroup with two methyl groups and a dodecyl aliphatic chain. The differences in vesicle morphologies (Figures 2, 4, 6, and 7) between the amphiphiles may be used to correlate the affect of molecular structures on self-aggregating properties. Derivative VI has a molecular structure similar to other amphiphiles known to form bilayer vesicles, (e.g., synthetic surfactants with two long alkyl chains and one headgroup).26-28 However, the molecular structure of derivative VII is unique, and hence, its vesicle-forming properties were difficult to predict. This difference in vesicle morphology as seen in the TEMs of derivatives VI and VII and their ability to form encapsulating vesicles may be rationalized using the intrinsic curvature (R0) hypothesis derived for lipid-polymorphism relationships.29 In this hypothesis, R0 is an amphiphile characteristic related to the mono- or bilayer curvature of the amphiphile aggregates that minimizes its elastic free energy.30 Typical lyotropic phase structures are spherical micelles, nonspherical micelles (H1), planar bilayer L∞, inverted hexagonal phases (H11), and inverted micelles. Changes in the molecular parameters of the amphiphiles, and hence in their R0, can be used to predict changes in the lipid phase of these amphiphiles. For example, increasing headgroup areas will increase R0, whereas increasing hydrophobic volumes will decrease R0. Addition of derivatives such as cholesterol would increase the hydrophobic domain volume as compared to the headgroup area promoting the transformation from planar bilayer (L∞) to inverse micelle phase (H11) or micelle (H1) to planar bilayer (L∞) phase. Alternatively, increasing headgroup size will increase R0, which would increase micelles (H1) vs planar bilayer. The above-described hypothesis may be used to estimate the effect of changes in molecular structures between derivatives VI and VII on their respective intrinsic (26) Okahata, Y.; Kunitake, T. Ber. Bunsen-Ges. Phys. Chem. 1980, 84, 550. (27) Nagamura, T.; Mikara, S.; Okahata, Y.; Kunitake, T.; Matsuo, T. Ber. Bunsen-Ges. Phys. Chem. 1978, 82, 1093. (28) Kunitale, T.; Okahata, Y. Bull. Chem. Soc. Jpn 1978, 51, 1877. (29) Kirk, G. L.; Gruner, S. M.; Stein, D. L. Biochemistry 1984, 23, 1093. (30) Gruner, S. M. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 3665.
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Figure 10. GFP expressing COS-7 cells after transfection with GFP-encoding cDNA COS-7 cells were grown in 96-well plates (10 000 cells/well) and exposed to equal amount of cDNA (4.4 µg/cm2), either encapsulated in vesicles or complexed with DEAE-dextran. The number of GFP expressing cells per field was counted using a fluorescent microscope with a 20× objective.
curvature and phase formation. Without additives such as cholesterol, derivative VI can form vesicles in DI water (these vesicles were shown to encapsulate uranyl acetate). Derivative VII appears to form smaller spherical structures (Figure 4a,b) under similar conditions that do not encapsulate uranyl acetate. The fact that amphiphile VII can form vesicles that do encapsulate uranyl acetate upon the addition of cholesterol, indicates that the structures formed without cholesterol are spherical and nonspherical micelle phases (H1). Thus, when three vernolic acid derivative structures are bound together as in derivative VII, the resulting headgroup area of the amphiphile is larger than the combined cross-sectional area of the aliphatic chains. With derivative VI, however, the formation of encapsulating spherical vesicle structures would indicate that with only one vernolic acid derivative structure the head and aliphatic chain cross-sectional areas of this amphiphile are similar. In effect, the molecular parameter areas of derivative VII are not a linear additive of the derivative VI. The intrinsic radius of curvature of derivative VI would thus be closer to forming planar phases, whereas that of derivative VII would form micelle phases in DI water. When vesicles of derivative VII are formed in aqueous solutions containing salts such as tris saline buffer, larger structures of up to 400 nm are formed (Figure 4c). With uranyl acetate in the solution, the internal structure of these vesicles appears to include fibers and ribbons (Figure 6) which may be manifestations of the H1 phase between spherical micelles and planar bilayers. The homogeneous particle population of 30-40 nm vesicles shown in Figure 4b was prepared with derivative VIIb without cholesterol. It is postulated that cationic headgroups are present on the surface of the particle, whereas the glycerol backbone of this amphiphile may be expected to face the interior of the nanoparticle and to be self-associated via polar interactions in the particle core. The presence of hydroxyl hydrogen bonding on the surface of the particle may add to the overall stability. NMR spectra (Figure 9) show that this derivative has a more rigid headgroup setting than the methyl-ester derivative. The DLS analysis over a period of 28 days also indicated that the structure of the nanoparticles made from the quaternary vernonia oil derivative is stable. Gene Transfection. Vesicles made from the quaternary vernonia oil derivative VIIb were found to be efficient in transfection of cDNA encoding for GFP into cultured COS-7 cells. The transfection efficiency was dependent on the concentration of the amphiphilic derivative used for the vesicle formation. When the concentration of the amphiphilic derivative was increased from 5 to 10 mg/ mL, the transfection efficiency was almost doubled (Figure 10). For 10 mg/mL, the molar ratio amphiphile to DNA
Derivatives from Vernonia Oil
Figure 11. Effect of the amount of cDNA on transfection efficiency. COS-7 cells were exposed to 2.2 or 4.4 µg/cm2 cDNA either encapsulated in VIIb derived vesicles or complexed by DEAE-dextran. The number of GFP expressing cells per field was counted using a fluorescent microscope with a 20× objective.
base pairs was approximately 1000 and the DNA itself was a 4.2Kb plasmid. The percent of transfected cells obtained with cDNA encapsulated in vesicles was higher than that obtained by the reference transfection technique with DEAE-dextran (Figure 10). The higher transfection efficiency with VIIb-derived vesicles was also expressed in terms of the amount of cDNA needed for transfection; that is, with vesicles, less cDNA yielded more transfected cells than did a larger amount of cDNA complexed with DEAE-dextran (Figure 11). In addition, the cationic vesicles provided protection to the cDNA against DNase I compared to DEAE-dextran. Transfection of cDNA of the GFP reporter gene, complexed with DEAE-dextran, was almost annulled after 20 min exposure to DNase I, whereas a significant number of transfected cells were still obtained after 20 min exposure of the cDNA containing vesicles to DNase I. It is thus suggested that some of the complexed cDNA was encapsulated and was not accessible to DNase I activity but may still be transferred into the cell, probably by fusion of the vesicle membrane with the cell membrane. These results show that the derivative that contains three cationic charges, each on a different aliphatic chain, connected via a glycerol moiety, can form vesicles that can be used as an effective vector for gene transfection. Conclusions The two quaternary amphiphilic derivatives synthesized from vernonia oil were shown to have vesicle-forming
Langmuir, Vol. 21, No. 17, 2005 7645
properties. Both amphiphiles are structurally related: derivative VII is essentially three molecules of derivative VI, bound together through a glycerol moiety. It was shown that the triple-headed derivative did not form encapsulating vesicles unless cholesterol was added, whereas the single-headed derivative, having two aliphatic chains, formed vesicles that encapsulated a marker without the addition of cholesterol. From the TEMs and the different abilities of the derivatives to encapsulate uranyl acetate, it was estimated that in amphiphile VI the headgroups and aliphatic chain cross-sectional areas were similar, allowing bilayer and spherical vesicle formation. The amphiphile VII, however, had a higher cross-sectional headgroup area than the aliphatic chain and formed both spherical and nonspherical micelle structures. By adding cholesterol to the formulation, the aliphatic chain crosssectional areas approached the dimensions of the headgroup areas and spherical vesicles could have been form. Vesicles prepared from the triple-headed cationic derivative VII proved to have good gene transfection properties. These properties were in accordance with the high density of the cationic groups, multiple hydrophobic chains, and the presence of the glycerol moiety at the opposite end of the molecule, a combination that facilitated the complexation of DNA and its transport across biological barriers. The cDNA thatwas encapsulated in the vesicles seems to be protected from hydrolysis by DNase. This derivative and similar molecules are currently under investigation for their DNA transfection properties. Acknowledgment. This study was supported by the Seed Money Fund of the Dean for Research & Development and the Applied Research Fund for Biotechnology Research of BG Negev Technologies Ltd., Ben-Gurion University of the Negev. We thank Ms Dorot Imber and Ms. Inez Mureinik for editing the manuscript. Supporting Information Available: Experimental procedures detailing the synthesis of haloacetate derivatives of methyl vernolate (IVa and IVb) and of vernonia oil (Va and Vb). This material is available free of charge via the Internet at http://pubs.acs.org. LA050091J
