Bioconjugate Chem. 2006, 17, 15−20
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Unilamellar Liposomes with Enhanced Boron Content Tiejun Li, Julie Hamdi, and M. Frederick Hawthorne* Department of Chemistry and Biochemistry, University of California, Los Angeles, 607 Charles E. Young Drive East, Los Angeles, California 90095. Received May 5, 2005; Revised Manuscript Received October 20, 2005
A new type of boron-rich, DSPC-free, unilamellar liposomes was formed using the novel dual-chain, ionic, nidocarborane lipid, K[nido-7-(C16H33OCH2)2CHOCH2-7,8-C2B9H11] (DAC-16), and cholesterol for encapsulation of an aqueous buffer core. Since DSPC was not necessary for the formation of stable DAC-16 liposomes, the boron concentration of these vesicles was increased dramatically to approximately 8.8 wt % in the dry lipid; these liposomes had a high bilayer boron incorporation efficiency of 98%. DSPC-free liposomes exhibited a size distribution pattern of 40-60 nm, which was in the range normally associated with selective tumor uptake. This size distribution was maintained throughout storage at room temperature for several months. Additionally, optimized liposome formulations incorporating DAC-16, DSPC, and cholesterol were identified having stable size distribution patterns after storage for more than two months at a variety of temperatures. Although animal studies indicate that DAC-16 liposomes are toxic, this new ionic nido-carborane lipid allows the formation of liposomes of high boron content for in vitro applications that require the delivery of large amounts of boron.
INTRODUCTION Unilamellar liposomes have been studied as drug delivery agents for more than 30 years because of their many unique properties, most notably the ability to selectively deliver large quantities of a wide variety of encapsulated agents to tumor sites (1, 2). Rapidly growing tumors have leaky vasculature and immature capillary endothelia through which small liposomes ( 5 are very hydrophobic and will be located almost exclusively in the lipid bilayer. Amphiphilic molecules, with log Poct between 1.7 and 4, have lower bilayer incorporation efficiencies than lipophilic species, and they are more easily lost from the bilayer because their biphasic nature allows for rapid transfer between the lipid and aqueous phases. The partition coefficients of 2, 1, DSPC, and phenylboronic acid were determined by measuring the concentration of boron or phosphorus present in each of the 1-octanol and water layers after equilibrium was reached. The previously studied phenylboronic acid (37) was included to validate the method when using ICP-AES boron analyses. As expected, the results (Table 1) indicated that 2 (log P ) 1.282) was more hydrophobic than 1 (log P ) 1.069), while less lipophilic than DSPC (log P ) 1.673). Because of its increased hydrophobic character, lipids incorporating 2 are expected to do so with higher efficiency than lipids incorporating 1. After formation of the liposomes by sonication, the unincorporated DAC-16 was separated from the lipid mixture through centrifugal filtration with Centricon membranes. The incorporation efficiency for DSPC-free DAC-16 liposomes (1:1 ratio of 2:cholesterol) was determined by ICP-AES analysis of the unincorporated DAC-16 in the filtrate and was found to be 98% for the DSPC-free liposomes, compared to the previously observed (18) average incorporation efficiency for 1 of 70% in the optimized formulation. This remarkably high incorporation efficiency indicates that, in the absence of DSPC, almost all of the DAC-16 carborane derivative is incorporated by the bilayer membrane of the liposomes. Because of applications in drug delivery the long-term stability of liposomes is another important property for consid-
Bioconjugate Chem., Vol. 17, No. 1, 2006 19
Unilamellar Liposomes with Enhanced Boron Content Table 2. Size Distribution Data of DSPC-free Liposomes (1:1 ratio of 2:cholesterol) storage temp
24 hours
1 week
2 weeks
4 weeks
2 months
23 °C 4 °C
38.5a -
39.3 40.4
42.3 38.1
38.9 37.5
39.8 40.1
a
mv values reported in nm.
Table 3. Preliminary in Vivo Evaluation of Liposomes Containing 2 liposome composition (DSPC:2:CH)
mv (nm)
%B in bilayer (dry lipid)
0:1:1
40.1
8.8
1:1:2
60.9
4.2
1:2:3
56.1
5.7
1:1:2a
56.4
4.3
1:0:1
55.4
-
B concnb of injected suspension (ppm)
injected dose (mg B/kg mouse)
mortality (hours until mouse death)
1525 763 750 325 1000 700 300 750 325 -
30 15 15 6.0 20 14 6.0 15 6.5 -
24 24 24 24 24 24 24 48c 48c -
a
Sodium salt of 2 was employed rather than the corresponding potassium salt. b Serial dilution of the concentrated liposome suspension was used to obtain the diluted samples. c Mice appeared to be in shock after 24 h.
eration, the ideal liposomes must have a long shelf life throughout which structural integrity is maintained. The stability of liposomes incorporating 2 into DSPC/cholesterol vesicles was tested by storing two different samples for two months, one at room temperature and the other at 4 °C. The size distribution pattern of the two samples was monitored frequently throughout this time interval, with no obvious changes in vesicle diameters (mv) observed during this period (Table 2). Studies of 2 in Vivo. Liposomes formed from nido-carborane, 2, have a much higher boron content than liposomes incorporating nido-carborane, 1; the enhanced incorporation efficiency of 2 into lipid bilayers is likely due to the increased hydrophobicity of the DAC-16 lipid. The increased boron content resulting from incorporation of 2 could also affect the biological behavior of the consequent liposomes. The biodistribution studies of such liposomes, containing 2, DSPC, and cholesterol in varied proportions, were performed at Washington State University using male BALB/c mice (about 10 g body weight) bearing small EMT-6 tumors. Typically 200 µL of each liposome suspension was injected into the tail vein, and the behavior of the mice was followed for up to 48 h. Unfortunately, in each case, the liposomes were found to be very toxic: no mouse survived longer than 48 h following injection of doses ranging from 6 to 30 mg of boron per kg of body weight. Since high potassium ion concentrations present in 2 could have been the cause of the observed toxicity, one liposome formulation contained the sodium salt of 2, but these liposomes also proved to be lethal (Table 3). A control formulation, containing only DSPC and cholesterol, clearly proved that the toxicity was attributable to 2, since mice injected with the control liposomes showed no adverse effects well past 48 h. Because of this result, further biodistribution tests were halted and no data were obtained on DAC-16 tumor boron concentration or tumor:blood ratio prior to animal death. For evaluation purposes, this information was compared to previously unpublished biodistribution data (38) obtained for the free uncomplexed DAC-16 compound, performed under similar conditions as reported above for the lipidic DAC-16. These results indicated acceptable tumor boron uptake with good tumor:blood ratios early in the trial period. Tumor boron values steadily increased over the course of the study, reaching 4.8 µg of B per gram of tumor tissue 50 h postinjection with a tumor: blood ratio of greater than 3 upon injected dose of 61 µg of B.
Though the free DAC-16 did not indicate the same degree of toxicity seen for the compound in the complexed lipidic-version, all BALB/c mice involved in the study were clearly affected and barely survived. Attempts to minimize this toxicity are under investigation. Although liposomes containing DAC-16 cannot be used as boron delivery vehicles for BNCT, the use of DAC16 does provide a method of preparing liposomes of high boron content that may be suitable for other applications.
CONCLUSION A new family of liposomes is reported here, assembled with DSPC, cholesterol, and a novel dual-chain nido-carborane amphiphilic lipid of high boron content, 2. The resulting small liposomes exhibit satisfactory stability and size distribution with mv in the range of 40-60 nm. Also, the new lipid component exhibited very high bilayer incorporation efficiency (98%) during liposome formation. A DSPC-free liposome was produced when using a lipid mixture containing only equimolar ratios of 2 and cholesterol. Because DSPC was not required, the boron content of the resulting liposomes is markedly increased compared to previously synthesized DSPC-containing liposomes (9, 18). Although the animal studies showed liposomes derived from 2 to be too toxic for use in BNCT, these liposomes contain a new type of bilayer constituent of high boron content, and they may prove to be suitable for applications involving boron delivery in vitro.
ACKNOWLEDGMENT This research was performed under the auspices of the Department of Energy (DOE) under contract DE-FG0395ER61975 with the additional assistance of a grant-in-aid from U.S. Borax, Inc. Support for equipment was provided by the National Science Foundation under equipment grant numbers CHE-9974928 and CHE-0092036. Supporting Information Available: Full experimental details and characterization data. This material is available free of charge via the Internet at http://pubs.acs.org.
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