Liposomes are vesicular systems that—among other applications—are used to deliver drugs for treating cer-tain cancers and microbial infections. Currently, drug delivery with liposomes is accomplished by a passive targeting approach, which relies on an acceptable circu-latory lifetime of the liposomes and their access to the diseased sites via vascular porosity (Lasic, 1998). Active targeting to a diseased site requires a combination of liposome stability, penetrability through biological bar-riers, and a controlled disruption mechanism to release the drug at the target site. A delivery system that has all these characteristics is difficult to achieve, particularly with cationic liposomes, which penetrate well biological barriers but, generally, have low stability in biological environments and are cleared rapidly by the liver and
lung (Litzinger et al., 1996). Extensive work has been carried out on active targeting, focusing on phospholipid amphiphilic compounds with a single head group that form bilayer membranes (Ansell et al., 2000; Metselaar et al., 2002; Sofou, 2007; Huwyler et al., 2008). On the other hand, bolaamphiphilic compounds, with two head groups at both ends of a hydrophobic domain, may form monolayer membrane vesicles with the potential advan-tage of having improved mechanical and physical stabil-ity, as was shown for archaeosomes (Patel and Sprott, 1999). The high stability of monolayer membranes made from bolaamphiphiles is partly due to the minimiza-tion of fusion that results from reduced lipid exchange because of the high activation barrier against pulling the inner charged head group through a hydrophobic matrix of a monolayer membrane (Fuhrhop and Mathieu, 1983; Fuhrhop and Wang, 2004). This feature of monolayer
Journal of Liposome ResearchJournal of Liposome Research, 2010; 20(2): 147–159
2010
Address for Correspondence: Eliahu Heldman, Department of Clinical Biochemistry, Ben-Gurion University, P.O. Box 653, Bergman Campus, Building N2, Beer Sheva 84105, Israel; Tel: (+)972-8-647-2969. E-mail: [email protected]
Cationic vesicles from novel bolaamphiphilic compounds
Mary Popov1, Charles Linder2, Richard J. Deckelbaum3, Sarina Grinberg4, Inge H. Hansen3, Eleonora Shaubi4, Tal Waner1, and Eliahu Heldman1
1Department of Clinical Biochemistry, Ben-Gurion University, Beer Sheva, Israel, 2Department of Biotechnology Engineering, Ben-Gurion University, Beer Sheva, Israel, 3Institute of Human Nutrition, Columbia University Medical Center, New York, New York, USA, and 4Department of Chemistry, Ben-Gurion University, Beer Sheva, Israel
AbstractEffective targeted drug delivery by cationic liposomes is difficult to achieve because of their rapid clear-ance from the blood circulation. Bolaamphiphiles that form monolayer membrane may provide vesicles with improved stability, as shown for archaeosomes. We investigated a series of bolaamphiphiles with ace-tylcholine head groups and systematic structural changes in their hydrophobic domain for their ability to form stable nanovesicles. Bolaamphiphiles with two aliphatic chains separated by a short amide midsection produced spherical nanovesicles ranging in diameter from 80 to 120 nm. These vesicles lost their encapsu-lated material within 24 hours of incubation in phosphate-buffered saline (PBS). Similar bolaamphiphiles with a longer midsection produced a mixture of fibers and more stable nanovesicles. Bolaamphiphiles with ester amide midsection produced only spherical nanovesicles that were stable during incubation in PBS for several days. Vesicles made from bolaamphiphiles with acetylcholine head groups conjugated to the aliphatic chain via the amine were less stable than vesicles made from bolaamphiphiles with head groups conjugated to the aliphatic chain via the acetyl group. Vesicles that were stable in vitro showed good stabil-ity in the blood circulation after intravenous administration to mice. These results help in elucidating the bolaamphiphile structures needed to form stable cationic vesicles for targeted drug delivery.
Keywords: Bolaamphiphiles; vesicles; liposomes; monolayer membrane; drug delivery
membrane may allow such vesicles to penetrate intact through biological barriers without fusing with the membrane while passing through it. Cationic vesicles with monolayer membrane may be particularly useful in penetrating cell membrane intact, as it has been shown that particles with cationic surfaces penetrate through biological barriers, such as the blood-brain barrier, via transcytosis (Fenart et al., 1999). Therefore, it is possible that cationic liposomes may serve as a vehicle system to transport drugs into the brain. However, cationic lipo-somes suffer from rapid clearance from the blood circu-lation, in part due to aggregation, and, therefore, manip-ulation of their surface is required to extend circulatory lifetime (Rejman et al., 2004). Cationic liposomes and cationic phospholipid complexants of polynucleotides are extensively studied (Templeton, 2003), and it has been shown that the problem of rapid clearance may be overcome, to some extent, by using a combination of amphiphilic compounds that reduce the overall cationic charge on the liposome’s surface (Fletcher et al., 2006). Reducing the cationic surface charge of liposomes, how-ever, may reduce their penetrability via biological barri-ers. On the other hand, cationic monolayer membrane vesicles made from bolaamphiphiles may have a longer circulatory lifetime together with good permeability via biological barriers, because these vesicles combine the cationic surface groups that enhance penetration with the reduced fusion characteristic that allow the vesicle to cross intact the cell membrane.
In the present study, we used novel bolaamphiphilic compounds with acetylcholine (ACh) head groups that formed cationic monolayer vesicles. ACh was chosen as a head group for its ability of producing cationic vesicles with a potentially selective disruption mechanism. The selectivity is based on head-group hydrolysis at sites with acetylcholine esterase (AChE) activity, where, upon removal of the head group, the vesicular structure dis-rupts and the encapsulated material is released (Menger and Johnston, 1991). Based on this concept, we proposed that vesicles formed from bolaamphiphiles with ACh head groups will also release the encapsulated material upon removal of the head groups by cholinesterases. However, elements that increase vesicle stability may be required when bolaamphiphiles are used for the forma-tion of vesicles. Vesicle stability may be increased by the introduction of stabilizing polar or hydrogen-bonding moieties within the hydrophobic domain, and/or within the interface between the hydrophilic and the hydropho-bic domains of the bolaamphiphile. The synthesis of such bolaamphiphiles can be simplified by using, as the start-ing material, vernolate (cis-12,13 epoxy,cis-9 octadecenoic acid) (Grinberg et al, 1994) (Figure 1), the main constitu-ent of vernonia oil. The epoxy group in the C12–C13 posi-tion of vernolic acid is used to introduce the head groups as well as the hydroxyl group—a hydrogen bond forming
groups, in a position that allows the modification of the interface region between the head groups and the alkyl chain (Grinberg et al., 2005). Reaction of vernonia oil with aliphatic diamine or diols results in the formation of the bolaamphiphilic skeleton with two vernolic acid chains coupled to each other via a diamide or ester midsection without disturbing the epoxy group (Grinberg et al., 2008). Thus, hydrogen bonding and polar groups can be readily incorporated into the aliphatic chains of variable lengths to form a variety of bolaamphiphilic compounds. The ability of such bolaamphiphilic compounds to form mon-olayer membrane cationic nanosized vesicles and the molecular parameters needed to confer stability on such cationic vesicles were investigated in the present study.
Materials and methods
Materials
Vernonia oil, containing an average of 2.1 epoxy groups per molecule of oil, was purchased from Ver-Tech, Inc. (Bethesda, Maryland, USA). 3H-cholesteryl oleyl ether ([1 alpha, 2 alpha (n)-3H]Cholesteryl oleyl ether, 30–60 Ci/mmol) was purchased from Amersham Biosciences, Inc. (UK), Carboxyfluorescein, Triton X-100, and 2,2,2-tribromoethanol (Avertin) were purchased from Sigma Chemicals (Israel). The following physiological solutions were prepared in our laboratory: physiological saline (0.9% NaCl) and phosphate-buffered saline (PBS; pH 7.4), containing 8 g of NaCl, 0.2 g of KC1, 1.44 g of Na
2HPO
4,
and 0.24 g of KH2PO
4 per liter. Chromatographic purifi-
cation of vesicle samples was carried out on a CL-2B col-umn for gel permeation chromatography (GPC), using Sepharose CL-2B (Amersham Biosciences, UK) and the eluent was lipoprotein buffer (LPB), containing 150 mM of NaCl, 0.24 mM of ethylene diamine tetraacetic acid (EDTA), and 2 U/mL of heparin.
Procedure for the synthesis of bolaamphiphiles
Synthesis of diglutarate derivativesA mixture containing 8.3 mmol of diamide or diester of vernolic acid (Grinberg et al., 2008) and 125 mmol of glutaric acid in 50 mL of 1,2 dichloroethane was refluxed for 48 hours. After cooling, 200 mL of 1,2 dichloroethane were added, and the reaction mixture was washed with a saturated NaCl solution until pH = 6 was obtained. The organic phase was dried over magnesium sulfate, filtered, and the solvent was removed under reduced pressure. Purification of the crude product was per-formed by column chromatography on silica-gel 60, using hexane:ether:acetic acid (5:5:0.1) as the eluent. This procedure gave a yield of about 42% of the product (III in Figure 2) with 98% purity.
Synthesis of di(N,N-dimethyl amino ethanol)diglutarateA solution of 0.054 mol of 1-ethyl-3-(3dimethylamino-propyl)carbodiimide (EDCI) in 100 mL of dry dichlo-romethane was added dropwise to an ice-cooled solution of 50 mL of dry dichloromethane containing 0.013 mol of compound III, 0.072 mol of 4-dimethyl-aminopyridine (DMAP), and 0.16 mol of N,N-dimethyl amino ethanol. The reaction mixture was stirred for 2 days at room temperature. The organic solution was washed several times with a saturated solution of NaCl until pH = 7 was obtained. The organic phase was dried over anhydrous MgSO
4, and the solvent was removed
under reduced pressure. The crude product was puri-fied by flash column chromatography with acetone as the eluent, and the purity of compound IV (Figure 2) was determined by high-performance liquid chroma-tography (HPLC) (methanol:water containing 0.15% trifluoroacetic acid; 95:5).
The following data were obtained for each of the syn-thesized compounds:Product IV (Z = NH; n = 2): 1H-NMR (500 MHz, CDCl
Synthesis of the bolaamphiphilesA mixture of compound IV and 1 mL of CH
3I in 20 mL of
dry dichloromethane was stirred for 24 hours in a cooling bath. The solvent was removed under reduced pressure, and the iodide was exchanged with chloride on an ion-exchange resin (Amberlite CG-400-I) purchased from Sigma-Aldrich (Isreal) to yield product V (Figure 2).
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Cationic vesicles from novel bolaamphiphilic compounds 151
The following data were obtained for each of the syn-thesized compounds:GLH-5 (Z = NH; n = 2): 1H-NMR (500MHz, DMSO-d
ESI–MS/MS (positive mode) m/z: [M+]/2 = 583.6 (z = 2).FT-IR (neat, cm−1) 3413, 1735, 1726.Elemental analysis: argentometric titration calculated for C
66H
122N
2O
4Cl
2, Cl− 5.7%; found Cl− 5.6%.
Preparation and characterization of vesicles
Vesicle preparationVesicles were prepared from the bolaamphiphiles by ethanol injection or film hydration, followed by bath sonication (New, 1993; Torchilin and Weissig, 2003) for 1 hour at 40°C in PBS or in 0.9% NaCl with a final bolaam-phiphile concentration of 10 mg/mL. Briefly, when vesicles were prepared by film hydration and sonica-tion, the bolaamphiphile was dissolved in chloroform/methanol and a film form under vacuum was a rotary evaporator. After leaving the film under vacuum over-night, it was then hydrated in PBS or 0.9% NaCl, followed by bath sonication. When the vesicles were prepared by ethanol injection, the bolaamphiphile was dissolved in ethanol:water (95:5) and injected into a stirred PBS solu-tion to form a 10-mg/mL suspension of amphiphile. The suspension was then subjected to bath sonication, as described above. For the encapsulation of carboxyfluo-rescein (CF), vesicles were prepared as described above, but with the addition of 0.1 mg/mL of CF to the PBS.
For the preparation of labeled vesicles, the marker, 3Hcholesteroyl oleoyl ether, was added to the amphiphile mixture (75 µL of 1 mCi/mL was added to 10 mg of bolaamphiphile in 3 mL of PBS), prior to a probe soni-cation. Labeled vesicles were stored under argon in a cold room. To determine particle size and homogene-ity, 500 µL of each sample was passed through a CL-2B column. In a typical experiment, to prepare labeled vesicles, 10 mg of the relevant derivative were dissolved in chloroform:methanol (2:1), containing 75 µL of 3Hcholesteryl oleoyl ether. The solution was dried under nitrogen for 30 minutes and then placed in a vacuum desiccator overnight to remove any residual solvent. The dried sample was hydrated by adding 3 mL of 0.9% NaCl and then vortexed and sonicated for 1 hour at 40°C under a stream of nitrogen. To remove residual Ti particles released from the probe during sonication, the sample was spun for 3 minutes at 3,000 rpm (800 rcf). Finally, the vesicle preparation was stored under argon at 4°C.
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152 M. Popov et al.
Determination of vesicle size, homogeneity, and morphologyThe vesicles were characterized with respect to their homogeneity by chromatography on a Sepharose CL-2B column and for their size and morphology by dynamic light scattering (DLS) and transmission electron spec-troscopy (TEM). The CL-2B column was prepared prior to sample application by washing it with a continuous flow of degassed LPB for 5 hours. For the chromatog-raphy, 500 µL of vesicle sample was applied to the col-umn, followed by washing with PBS, which was added continuously while fractions of 1.25 mL of the effluent were collected and 625 µL counted from each fraction. To obtain the mean particle size of the vesicles, DLS measurements were performed in an ALV-CGS-3 (ALV, Langen, Germany) spectrometer at 25°C and at a wave-length 632.8 nm, with a scattering angle of 90 degrees. TEM was performed with a Jeol JEM-1230 TEM (JEOL Ltd., Tokyo, Japan) at 80 kV, and electron micrographs were taken with a TemCam-F214 camera [Tietz Video and Image Processing Systems (TVIPS), Gauting, Germany]. For vesicle visualization by TEM, vesicle sus-pension (10 µL) was spread on a 300 mesh copper grid coated with a Formvar film, and, after 30 seconds, the excess fluid was removed by a filter paper. Then, 10 µL of uranyl acetate (1%) was added to the grid for another 30 seconds and the excess fluid removed. The grid was then dried in the air for about 3 hours and placed in the TEM.
Determination of apparent encapsulation efficiency and vesicle stabilityQuantitative measurement of encapsulation and evalu-ation of vesicle stability in vitro were performed by using carboxyfluorescein (CF) as the labeled encapsulated marker. The solution of vesicles prepared in the pres-ence of 0.1 mg/mL of CF was diluted (1:200) in PBS (pH 7.5), in order to reach the linear fluorescence range of the dye, and an aliquot of 3.5 mL was transferred into a quartz cuvette for the measurement of fluorescence at an excitation wavelength of 492 nm and an emission wavelength of 517 nm (employing the PMT 450 kinetics program) in a Varian Cary Eclipse spectrofluorimeter (Varian Inc. Walnut Creek, California). After measuring the initial fluorescence, Triton X-100, at a final con-centration of 0.15%, was added to the cuvette, and the fluorescence was measured again. The apparent encap-sulation efficiency was then calculated, according to the following equation (New, 1993):
R R
REncapsulationAf B
Af
−× =100 %
where RB is the initial fluorescence reading and R
Af is the
fluorescence reading after the addition of Triton X-100.
To determine vesicle stability, samples of the vesicles with encapsulated CF were stored in PBS at either 4°C or at room temperature, and the percent of apparent encapsulation was determined at different times of storage.
In vivo studies
To determine blood clearance and organ distribution, 100 µL of a suspension containing radiolabeled vesi-cles was injected intravenously into 10-week-old male C57BL/6 mice (Jackson Laboratory, Bar Harbor, Maine) anesthetized with Avertin. Orbital blood was drawn at various times after the vesicle administration. Plasma clearance was calculated as detailed previously (Qi et al., 2002). To study distribution of the vesicles among various organs, mice were sacrificed 60 minutes after injection of the labeled vesicles, and various organs were dissected out and homogenized in PBS. Radioactivity in each organ was determined as described previously (Qi et al., 2002).
Results
Synthesis of bolaamphiphilic compounds
The synthetic strategy of the bolaamphiphilic com-pounds described here was based on: (1) exploiting the relatively low reactivity of the internal epoxy group of the vernonia moiety (Figure 1) to build the hydrophobic chain skeleton of the bolaamphiphiles (compound I in Figure 2). This was done by reacting the carboxylic group of the relevant vernonia moiety—vernonia oil, methyl vernolate, or vernolic acid—with aliphatic diamines or aliphatic diols to obtain bisvernolamides or bisver-nolesters, respectively. (2) The epoxy group, located on C12 and C13 of the aliphatic chain of vernolic acid, was used to introduce both the head group and the head group/aliphatic chain interface area moiety on the two vicinal carbons obtained after the opening of the oxirane ring. The pentyl pendant, attached to one of the vicinal carbons, provides the additional hydrophobic stabiliza-tion and geometric packing necessary to form spherical vesicles, rather than micelles or fibers.
The bolaamphiphiles—GLH-4, GLH-9 and GLH-20 (Figure 3)—contain ACh head groups attached to the vernonia skeleton (divernolamide or divernolester) through the nitrogen atom of the choline moiety. These bolaamphiphiles were prepared, as previously described (Grinberg et al., 2008), in a two-stage synthesis: First, opening of the epoxy ring with a haloacetic acid and, second, quaternization with the N,N-dimethylamino ethyl acetate. The bolaamphiphiles—GLH-5, GLH-10, and GLH-19 (Figure 3)—contain an ACh head group attached to the vernonia skeleton (divernolamide or
Cationic vesicles from novel bolaamphiphilic compounds 153
divernolester) through the acetyl group. These bolaam-phiphiles were prepared in a three-stage synthesis, including the opening of the epoxy ring with a dicar-boxylic acid (glutaric acid) and then esterification of the free carboxylic group with N,N-dimethyl amino ethanol (Figure 2). The final product is obtained by quaternization of the head group, using methyl iodide. The bolaamphiphiles—GLH-4, GLH-9, GLH-5, and GLH-10—have amide bonds within the hydrophobic domain, while the bolaamphiphiles—GLH-20 and GLH-19—have ester bonds within the hydrophobic domain (Figure 3).
Vesicle visualization by transmission electron microscopy (TEM)
TEM micrographs of vesicles made from each of the above six bolaamphiphiles are shown in Figure 4. In all cases, the size of the vesicles ranged between 50 and 100 nm. The bolaamphiphiles, GLH-4 and GLH-5,
which contain two aliphatic chains separated by a short amide midsection, formed spherical vesicles, whereas GLH-9 and GLH-10, which contain two similar aliphatic chains separated by a longer amide midsection, formed fibers, in addition to vesicles. The bolaamphiphiles, GLH-19 and GLH-20, with midsections of the same length as GLH-9 and GLH-10, but which contain ester groups rather than amide groups, formed only spheri-cal vesicles (Figure 4).
Determination of vesicle size by dynamic light scattering (DLS)
Analysis by DLS was carried out to determine size and homogeneity of the vesicles. Table 1 shows the diameter of the various vesicle formulations made by the film-hydration and sonication method. They show a major radii peak in the range of 40–70 nm. The sizes of the vesi-cles calculated from the DLS data are larger than those estimated from the TEM micrographs. This is expected,
C-(CH2)7-CH=CH-CH2-CH-CH-(CH2)4-CH3
O
(CH2)2
NH
HO O-CO-CH2-N-CH2-CH2-O-CO-CH3+
Cl–
C-(CH2)7-CH=CH-CH2-CH-CH-(CH2)4-CH3
O HO O-CO-CH2-N-CH2-CH2-O-CO-CH3
CH3H3C
CH3H3C
+Cl–
NH
GLH-4
C-(CH2)7-CH=CH-CH2-CH-CH-(CH2)4-CH3
O
(CH2)10
NH
HO O-CO-CH2-N-CH2-CH2-O-CO-CH3+
Cl–
C-(CH2)7-CH=CH-CH2-CH-CH-(CH2)4-CH3
O HO O-CO-CH2-N-CH2-CH2-O-CO-CH3
CH3H3C
CH3H3C
+Cl–
NH
GLH-9
C-(CH2)7-CH=CH-CH2-CH-CH-(CH2)4-CH3
O
(CH2)10
O
HO O-CO-CH2-N-CH2-CH2-O-CO-CH3+
Cl–
C-(CH2)7-CH=CH-CH2-CH-CH-(CH2)4-CH3
O HO O-CO-CH2-N-CH2-CH2-O-CO-CH3
CH3H3C
CH3H3C
+Cl–
O
GLH-20
GLH-5
(CH2)2
NH
NH
C-(CH2)7-CH=CH-CH2-CH-CH-(CH2)4-CH3
O HOCH3
CH3
CH3Cl–
O-CO-(CH2)3-CO-O-CH2-CH2-N+
C-(CH2)7-CH=CH-CH2-CH-CH-(CH2)4-CH3
O HOCH3
CH3
CH3Cl–
O-CO-(CH2)3-CO-O-CH2-CH2-N+
GLH-10
(CH2)10
NH
NH
C-(CH2)7-CH=CH-CH2-CH-CH-(CH2)4-CH3
O HOCH3
CH3
CH3Cl–
O-CO-(CH2)3-CO-O-CH2-CH2-N+
C-(CH2)7-CH=CH-CH2-CH-CH-(CH2)4-CH3
O HOCH3
CH3
CH3Cl–
O-CO-(CH2)3-CO-O-CH2-CH2-N+
GLH-19
(CH2)10
O
O
C-(CH2)7-CH=CH-CH2-CH-CH-(CH2)4-CH3
O HOCH3
CH3
CH3Cl–
O-CO-(CH2)3-CO-O-CH2-CH2-N+
C-(CH2)7-CH=CH-CH2-CH-CH-(CH2)4-CH3
O HOCH3
CH3
CH3Cl–
O-CO-(CH2)3-CO-O-CH2-CH2-N+
Figure 3. Chemical structures of bolaamphiphilic derivatives of vernonia oil containing ACh head groups.
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as the DLS measures the hydrodynamic radius, which includes the water of hydration around the head groups. In addition, the differences in the sizes, as determined by TEM vs. DLS, could also stem, in part, from the fact that DLS generally overestimates smaller particles in the presence of bigger ones, because the latter scatter more. Indeed, as can be seen from Figure 5, which shows a typi-cal DLS profile of vesicles made from GLH-20 and which corresponds to the sample used for studying the blood clearance and organ distribution in mice (described below), both a smaller peak, which corresponds to about 15–20 nm radius vesicles and a larger, broader peak with a radius of about 65 nm can be seen.
GLH-4 GLH-5
GLH-9 GLH-10
GLH-19 GLH-20
100 nm
100 nm
100 nm 100 nm
100 nm
100 nm
Figure 4. TEM micrographs of vesicles made from each of the six studied bolaamphiphiles. Vesicles were prepared by ethanol injection, followed by sonication.
Table 1. Radii of vesicles from various bolaamphiphiles measured by DLS.
Vesicles made from
Vesicles diameter (nm)
Relative peak width (nm)
Weight of peak (%)
GLH-4 84.0 ±9.7 95.31
GLH-5 64.4 ±7.6 89.39
GLH-4+GLH-5 (1:1)
116.5 ±11.4 94.56
GLH-9 123.9 ±15.7 97.84
GLH-19 88.3 ±13.6 77.47
GLH-20 84.9 ±8.25 94.72
Vesicles were prepared in PBS by ethanol injection, followed by sonication from 10 mg/mL of bolaamphiphile. Weight of peak represents percent of vesicles with the average size.
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Cationic vesicles from novel bolaamphiphilic compounds 155
Determination of vesicle homogeneity by gel permeation
The gel permeation elution profile on a CL-2B column of a typical GLH-20 vesicle preparation is shown in Figure 6. As can be seen, one major peak was eluted in the void volume. The size distribution of these vesicles corresponds to the major peak of the DLS profile shown in Figure 5, with an average radius of about 65 nm. A smaller peak, representing smaller vesicles, is also seen in the gel permeation chromatography profile (Figure 6), which corresponds to the small peak of 15–20 nm radius, seen in the DLS profile.
In vitro studies of vesicle stability
To compare the stability of vesicles made from the different bolaamphipihles that were examined in the present study, vesicles were stored at 4°C in the medium in which they were prepared (PBS with CF), and the percent of apparent encapsulation of CF was measured as a function of storage time. As can be seen in Figure 7, vesicles made from the amphiphile, GLH-4, which contains two aliphatic chains separated by a short amide midsection, are less stable than vesicles made from GLH-9, a bolaamphiphile similar to GLH-4, but with a longer amide midsection. Vesicles made from GLH-20, a bolaamphiphilic compound similar to GLH-9, but with an ester, rather than amide groups, in the midsection, were even more stable and did not loose their encapsulated CF, as long as they were not diluted.
Stability was also examined upon a 10-fold dilution in the medium in which the vesicles were prepared with-out CF (PBS). The results are shown in Figure 8, which compares vesicles from GLH-4 and GLH-5 and vesicles from GLH-19 and GLH-20, respectively. The stability of vesicles from GLH-4 and GLH-5 (Figure 8A) was exam-ined at 4°C, as these vesicles were found to be unstable at room temperature. By comparison, vesicles made from bolaamphiphiles with longer aliphatic chains and
ester midsections (GLH-19 and GLH-20), were more stable and, therefore, were tested at room temperature (Figure 8B). To compare the stability of vesicles with different degrees of apparent encapsulation, the data were normalized to represent the relative change in percent encapsulation, where the apparent encapsula-tion values at time zero were considered as 100%. The actual percent of apparent encapsulation in vesicles made from GLH-4, GLH-5, GLH-19, and GLH-20 were 20, 7, 18, and 25%, respectively. These stability studies show that the vesicles made from bolaamphiphiles with the ACh attached to the aliphatic chain via the acetyl group (GLH-5 and GLH-19) were more stable than the vesicles made from bolaamphiphiles with the acetylcholine head group attached directly through the
Figure 5. A DLS profile of vesicles made from GLH-20 by ethanol injection, followed by sonication, showing the DLS signal intensity versus size distribution.
0
300000
200000
100000
0 10 20 30 40 50 60 70 80Fraction number
Rad
ioac
tivel
y la
bele
d ve
sicl
es(C
PM
per
frac
tion)
Figure 6. Gel permeation chromatography profile of GLH-20 vesi-cles labeled with [3H]choleteryl oleyl ether on a CL-2B column. The eluent was lipoprotein buffer (LPB) containing 150 mM of NaCl, 0.24 mM of EDTA, and 2 U/mL of heparin. Fractions of 1.25 mL were collected and their radioactivity counted. Vesicles were made by film hydration, followed by sonication.
0 50 100 150 200 2500
20
40
60
80
100
120
Time (hours)
Per
cent
of i
nitia
l enc
apsu
latio
n
Figure 7. Stability of vesicles made from GLH-4, GLH-9, and GLH-20 at 4°C. Vesicles were prepared in PBS containing 0.1 mg/mL of CF by ethanol injection, followed by sonication, and stored in the medium in which they were prepared for various times. Percent encapsulation was determined at each time point. – GLH-4 vesilcles; – GLH-9 vesicles; – GLH-20 vesicles. Each point represents an average of data taken from four independent determinations ± standard deviation.
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amine (GLH-4 and GLH-20). Vesicles prepared from a mixture of the two bolaamphiphiles, GLH-4 with GLH-5 (Figure 8A), or GLH-19 with GLH-20 (Figure 8B), were also studied and demonstrated intermedi-ate stability, as compared to vesicles made from each bolaamphiphiles alone.
Determination of the vesicle life time in the blood circulation following intravenous administration
For the pharmacokinetic studies, we chose to work with vesicles made from GLH-20, as these vesicles showed good stability in vitro. Since cationic liposomes are known to be toxic, we tested the GLH-20 vesicles for toxicity in a preliminary experiment to determine a safe dose. These preliminary toxicity studies indicated that
the injection of 100 μL of vesicles made from 10 mg/mL GLH-20 did not cause toxic signs, and, therefore, this dose was chosen for the pharmacokinetic stud-ies. GLH-20 vesicles labeled with the nonhydrolyzable 3H-cholesteryl oleyl ether were injected into C57BL/6 mice, and the radioactivity in the blood was measured at various time points after the injection. The pharma-cokinetic profile of the labeled vesicles for the first hour is shown in Figure 9. As can be seen, there was an initial decline in blood levels, which was followed by a rise and then by a very slow decline in the content of the labeled vesicles in the blood. This phenomenon may indicate initial margination of vesicles at vessel walls (e.g., capillaries) with a subsequent release back into the circulation. Radiolabeled vesicles in blood circula-tion were examined also for longer times, and it was found that GLH-20 vesicles still have a blood level of about 15% at 24 hours after the administration of the vesicles.
Organ distribution
Organ distribution of vesicles made from GLH-20 was determined 60 minutes after the injection of the vesicles to mice. The organs studied were the heart, lung, liver, blood, kidney, spleen, muscle, bone, subcutaneous adipose tissue (SAT), and visceral adipose tissue (VAT) (Table 2). The organs with the highest radioactivity at 60 minutes after the injection were the blood (~42%) and liver (~48%) (Table 2). In preliminary studies with 2 ani-mals, we found that after 24 hours, radioactivity in the liver was the highest at close to 80% of the total counts (data not shown), indicating that the major pathway for vesicle elimination is hepatic.
Figure 8. Percent encapsulation as a function of incubation time at 4 (GLH-4 and GLH-5) and 25°C (GLH-19 and GLH-20), following a 10-fold dilution of the vesicles in PBS. Vesicles made by film hydra-tion followed by sonication in PBS containing 0.1 mg/mL CF from (a) GLH-4 (), GLH-5 (), and a mixture () of GLH-4 and GLH-5 (1:1) and (b) GLH-19 (), GLH-20 () and mixtures of GLH-19 and GLH-20 {1:1 () and 1:2 (). Percent encapsulation was normalized by using the initial encapsulation as 100%. Each point represents an average of data taken from three independent determinations ± standard deviation.
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Figure 9. Survival of GLH-20 vesicles in the blood circulation at vari-ous times after intravenous (i.v.) injection. Vesicles, labeled with [3H]choleteryl oleyl ether were prepared by film hydration, followed by probe sonication (the same vesicle preparation shown in Figures 5 and 6). Vesicles (50 µL) were injected i.v. into the tail of the mice. Orbital blood was withdrawn at the indicated time points and radio-activity determined.
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Cationic vesicles from novel bolaamphiphilic compounds 157
Discussion
To achieve targeted drug delivery by vesicles, impor-tant characteristics are penetrability through biological barriers, a selective disruption mechanism, and vesicle stability in the blood circulation to allow the vesicles to reach the target tissue while they are still intact. With the aim of preparing cationic vesicles with these charac-teristics, we synthesized and studied a number of novel bolaamphiphilic compounds with ACh head groups. Bolaamphiphiles from archaebacteria, which survive under extreme thermal and chemical conditions, are reported to form highly stable vesicles that may be supe-rior, compared to phospholipid liposomes, as a vehicle for drug delivery (Patel and Sprott, 1999). For example, it has been reported that bipolar tetraether bolaam-phiphiles form vesicles that have improved stability under extreme conditions (Chang, 1994). Cationic vesi-cles made from bolaamphiphiles, in addition to being stable, may penetrate via biological barriers (Fenart et al., 1999). Evidently, the vesicle stability is strongly influenced by the balance between the attractive forces of the aliphatic chains and the repulsive forces between the charged head groups. Removal of the head group will perturb this balance and will result in destabilization of the vesicular structure and release of the encapsu-lated material. Following these rationales, we designed the bolaamphiphiles so that they have enzymatically hydrolysable head groups (ACh head groups that may be hydrolyzed by cholinesterases). Before examining controlled decapsulation of the vesicles by enzymatic hydrolysis of the head groups, we investigated the novel bolaamphiphiles for their ability to form stable vesicles in vitro and in vivo.
The bolaamphiphile structure was shown to have a strong effect on the shape and the size of the vesi-cle, as seen in the TEM micrographs of the different
formulations (Figure 4). Spherical vesicles were read-ily formed from bolaamphiphiles with a short (CH
2)
2-
midsection and amide-connecting groups (GLH-4 and GLH–5). Lengthening the midsection [-(CH
2)
10-], as in
GLH-9 and GLH-10, resulted in the formation of both fibers and spherical particles, such that the solutions of these derivatives formed thixotropic gels at con-centrations as low as 0.1%. Remarkably, if esters were used instead of amide bonds in the midsection of these long-chain bolaamphiphiles (GLH-19 and GLH-20), then only spherical vesicles were formed, but not fib-ers. This finding suggests that bolaamphiphiles with an ester midsection may form more stable vesicles than bolaamphiphiles that interact by hydrogen-bonding via amides in the midsection. The presence of hydrogen bond-forming groups within the hydrophobic domain of amphiphilic compounds have been reported to induce fiber formation, rather than spherical vesicles (Fuhrhop and Mathieu, 1991), as, indeed, was seen also with the bolaamphiphiles with the amide midsection described in the present study. Symmetrical bolaam-phiphiles more often form fibers than vesciles (Fuhrhop and Wang, 2004). In our study, one of the successes was coming up with bolaamphiphiles, which form vesicles. The formation of bolaamphiphiles with alkyl pendants next to the head groups allowed the formation of spheri-cal vesicles when internal polar groups, such as esters, connecting the two aliphatic chains (GLH-19 and GLH-20). When we used amides within the aliphatic chain, the strong hydrogen bonding in the midsection of the alkyl chain of the bolaamphiphile (e.g., GLH-9) created conditions under which both fibers and vesicles were formed. The fibers are structures with both the amide and head groups in contact with the water on the surface of the self-aggregate structures. The spherical vesicle structures have the amide structures within the interior of a membrane comprising the hydrophobic alkyl chain, and the two structures are a result of a balance of the interfacial forces. We believe that in GLH-4, which has a short chain, the bolaamphiphile cannot form aggregate fiber because the alkyl chain pendants near the head group sterically interfere with the required curvature of the internal alkyl chains. By comparison, in the larger alkyl chain (e.g., GLH 9), the increased length of the alkyl midsection can curve into the configuration needed for fiber formation, in spite of the alkyl pendants. With esters instead of amides, the H-bonding interactions with water are significantly reduced and the lowest energy aggregate structures are spherical vesicles.
The actual percent encapsulation of CF, which were determined in the present study, were 20, 7, 18, and 25%, for vesicles from GLH-4, GLH-5, GLH-19, and GLH-20, respectively. Table 1 shows that the vesicle diameters vary in the range of 80–120 nm. Theoretical calculations, based on vesicle size and membrane thickness, can give
Table 2. Organ distribution of vesicles from GLH 20 after i.v. injection into mice.
OrganMean % ± SD (n = 4) of total vesicles found in all organs after 60 minutes
Heart 0.08 ± 0.02
Lung 3.69 ± 1.05
Liver 47.73 ± 2.20
Kidney 0.78 ± 0.12
Spleen 2.74 ± 0.81
SAT 0.10 ± 0.07
VAT 0.11 ± 0.13
Muscle 3.51 ± 0.35
Bone 0.19 ± 0.03
Blood 42.33 ± 3.22
Vesicles were injected to the same mice described in Figure 9. At the end of the blood-sample collection (60 minutes after the injection of the vesicles), animals were sacrificed, tissues dissected, homogenized in PBS (1 g of tissue per 4 mL of PBS) and radioactivity determined.
the inner volume of the vesicle core. This, together with the percent of the CF in the solution, gives an approxi-mate encapsulation of 5%. The much higher percent encapsulation that we measured in the vesicles is due to complexation of the anionic CF with the positively charged head groups. This is a known phenomenon (Wang et al., 2006) of association via complexation. In this context, it should be mentioned that the CF, which was used as a marker for encapsulation, does not neces-sarily represent encapsulation of a specific drug, and it has been used primarily to assess stability and to com-pare various vesicle preparations for their capability to encapsulate and bind negatively charged small mol-ecules. To assess the encapsulation of real drugs, condi-tions for each drug or agent have to be optimized. Thus, it is conceivable that each drug would have different encapsulation efficiencies and release kinetics. While the CF can be used as a model for anionic water-soluble, low-molecular-weight drug molecules, its function in this work was to determine the stability of the vesicles.
The estimated average vesicle size determined from the TEM is smaller for vesicles made from GLH-5, com-pared to vesicles made from GLH-4, where the ACh head group is attached than through the amine, rather than the acetyl, group. Consistently, the amount of the encapsulated material was larger with GLH-4 than that obtained with GLH-5 vesicles. This may be due to dif-ferences in packing parameters that should also affect vesicle stability. Indeed, vesicles that were made from GLH-4 were less stable than vesicles that were made from GLH-5. We propose that GLH-5 vesicles are more stable than GLH-4 vesicles due to more efficient packing in the head-group region, since the cationic nitrogen moiety in GLH-5 contains methyl groups, which are less bulky than the sterically larger acetyl group pendent of GLH-4. Yet, both of these vesicle preparations were less stable than vesicles made from bolaamphiphiles with long hydro-phobic chains, such as GLH-19 and GLH-20, indicating that elongation of the alkyl chain of the bolaamphiphile increases vesicle stability by virtue of stabilizing hydro-phobic interactions between the bolaamphiphiles that make the vesicle’s membrane.
Two mechanisms can lead to the loss of the encap-sulated material: disintegration of vesicles and diffu-sion of the dye out of the vesicles. The impact of vesicle disintegration deems to play the leading role there. A support to this conclusion comes from the findings that some of the vesicles (e.g., GLH-4 and, to a lesser extent, GLH-5) lost their encapsulated CF, even under condi-tions in which the intra- and extravesicular concentra-tions of the CF were equal (see Figure 7), so that a loss of encapsulated CF by diffusion out of the vesicle could not have occurred. Yet, the loss of the encapsulated material following dilution could be, in part, due to diffusion of the dye out of the vesicles because of the concentration
gradient, which was formed after the dilution of the vesi-cles. However, vesicle disintegration under these condi-tions is also possible, especially when bolaamphiphiles with high CMC were used to form the vesicles. Indeed, the vesicles made from the bolaamphiphiles with the short aliphatic chain (which should have higher CMC) lost the encapsulated CF upon dilution at a faster rate than vesicles made from bolaamphiphiles with a longer aliphatic chain (that should have lower CMC).
The similar stabilities of vesicles prepared from a combination of GLH-4 and GLH-5, and those formed from GLH-5 alone (Figure 8A), suggests that it is possi-ble to control vesicle stability by combining amphiphiles that form vesicles with different stabilities. The same argument could also be referred to vesicles made from a combination of GLH-19 and GLH-20 (Figure 8B).
We chose the GLH-20 vesicles to examine in vivo stability, since these vesicles were stable in vitro and their encapsulation capacity was high. From the calcu-lated fraction catabolic rates (FCRs), the blood removal for the GLH-20 vesicles was about 4 pools/hour. The relatively good stability of these vesicles in the blood circulation is unique for cationic particles, since it is generally accepted that cationic particles are rapidly cleared within minutes after intravenous administration if no stabilizing components were added to the vesicle surface (Fenart et al., 1999; Rejman et al., 2004; Thurston et al., 1998). Indeed, in contrast to sterically stabilized liposomes with good circulatory lifetimes (Huang et al., 1992), cationic liposomes are rapidly cleared by the lung, liver, and spleen (Thurston et al., 1998). However, in our study, blood levels of the cationic GLH-20 vesicles, with-out stabilizing surface groups, were still high 1 hour after the injection, indicating that bolaamphiphiles of the type used in the present study form vesicles with a relatively high biological stability. The decline in blood levels, fol-lowed by a slower decline, is typical for small liposomal particles, whereas larger particles are rapidly filtered. Notably, after injection, recovery in blood of the GLH-20 vesicles (estimated by extrapolation of recovered dose back to time zero) ranged between 51 and 62%.
The organ distribution of vesicles made from GLH-20 (Table 2) show high blood levels 60 minutes after the injection, with most of the organ uptake being by the liver (with some uptake by muscle and spleen). This biodistribution is similar to that of stealth liposomes, in spite of the fact that the vesicles, which were studied here, are cationic in nature and their lifetime in the blood is shorter. It is possible that the lifetime of the vesicles in the blood circulation will be extended if the choline esterases in the blood will be inhibited, since hydrolysis of the head groups by this group of enzymes, which are present in high quantities in the blood, reduce the cir-culatory lifetime due to a disintegration of the vesicles following the hydrolysis of the ACh head groups.
Cationic vesicles from novel bolaamphiphilic compounds 159
Finally, it should be mentioned that cationic lipo-somes are known to have relatively high toxicity, and, therefore, before the administration of the vesicles to animals, we carried out preliminary toxicity studies in order to determine a safe nontoxic dose for the phar-macokinetic studies. This preliminary experiment with the vesicles described in the present study showed that, for at least some of the vesicle preparation, a dose of 100 mg/kg did not cause apparent toxic signs.
Conclusions
In summary, the findings of this study validate our working hypothesis that bolaamphiphiles with ACh head groups form cationic vesicles with relatively good biological stability. The structure of such bolaam-phiphiles could be optimized to provide improved vesi-cle stability, which is needed for targeted drug delivery. Differences between vesicles prepared from various bolaamphiphiles may be attributed to aliphatic chain length and the different ACh head-group configurations, which allow for diverse packing of the bolaamphiphiles within the vesicle membrane. GLH-20 vesicles appear to have good circulatory stability for cationic particle, most probably due to the length of the hydrophobic chain, which promotes strong hydrophobic interactions and lowers the CAC, making the vesicle more resistant to disruption by dilution. The presence of hydrophilic ester groups in the midsection enables localized polar interactions between the bolaamphiphiles, which may further stabilize the vesicles. An approach based on the advantages conferred by the bolaamphiphilic compound—GLH-20—is now under development as a targeting release platform.
Acknowledgement
This study was supported by a grant from the United States–Israel Binational Science Foundation (No. 2003153).
Declaration of interest: The authors have declared no conflict of interest.
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