C O M M U N I C A T I O N S
We then added albumin into the OEPC liposomes at pH 5.2 and
8.5, respectively, and found that particles in the pH 5.2 buffer
immediately fell apart, while those in pH 8.5 buffer were
unchanged. On the basis of these observations, we believe that the
acid hydrolysis of OEPC liposomes ultimately leads to leaky
metastable vesicles consisting of lyso-PC and dodecanol. When the
amount of lyso-PC reaches a critical level, pores form on the surface
of the vesicle, resulting in rapid content release. Eventually, the
metastable vesicles can be completely destroyed when the lyso-
PC and dodecanol are removed from the bilayer by the albumin or
the interaction with other in vivo components.
OEPC is a new class ortho ester lipid with a biocompatible
phosphocholine headgroup. Highly pH-sensitive and biocompatible,
OEPC or its similar derivatives have great potential for applications
in drug and gene delivery. For example, when formulated with
cationic lipids, OEPC may facilitate the gene release from the acidic
endosome to the cytosol in a timely manner, minimizing the
lysosomal gene degradation. Our preliminary data showed that
OEPC significantly enhanced the in vitro transfection efficiency
compared with the pH-insensitive phosphocholine when formulated
with cationic lipids (8.24 × 105 versus 3.95 × 104 RLU/mg protein).
In summary, we have designed and synthesized a highly pH-
sensitive diortho ester phosphocholine (OEPC). The acid hydrolysis
of the OEPC liposome is fast and pH-dependent, and the liposomes
are transformed into leaky metastable vesicles that quickly collapsed
upon the interaction with albumin. The application of OEPC and
its derivatives on acid-triggered drug and gene delivery is promising.
Figure 1. The pH-dependent leakage of OEPC liposomes. Percentage of
leakage over time in glucuronate buffer (pH 4.5, 5.0, and 5.5) and phosphate
buffer (pH 6.0 and 7.0).
Acknowledgment. This work was supported by NIH EB003008
and GM61851. Z.H. is partially supported by the UCSF Cystic
Fibrosis Foundation. We dedicate this paper to Dr. Jorge Heller at
A.P. Pharma (Redwood City, CA) on his 79th birthday for the
generous gift of 3,9-diethylidene-2,4,8,10-tetraoxaspiro[5,5]undecane.
Thanks are also extended to the UCSF Mass Spectrometry Facility
(A.L. Burlingame, Director) supported by NIH NCRR RR01614.
Figure 2. Logarithm of lag time (second) of OEPC liposomes at various
pH. Linear regression for the data: log (lag time) ) -0.236 + 0.498 ×
pH, r ) 0.999, and P < 0.0001.
small portion of ANTS slowly leaks out, and a burst phase when
ANTS is quickly released. The lag time (the transition point from
the shallow slope to the steep slope)4,13 is pH-dependent;the lower
the pH, the shorter the time. In Figure 2, we plot the logarithm of
the lag time versus the buffer pH and observe a linear correlation
(r ) 0.999). The pH-dependent leakage of OEPC liposomes is very
similar to our previous observation on the POD liposomes but with
a relatively shorter lag time at the same pH. This difference may
come from the different destabilization mechanisms between the
OEPC liposomes and the POD liposomes.
For the POD liposomes, when the amount of surface POD is
lowered to a critical level, the lamellar-to-inverted hexagonal phase
transition is initiated in the phosphatidylethanolamine, and the
liposomes aggregate and collapse rapidly. As expected, the OEPC
liposomes do not spontaneously collapse under the acidic conditions.
Moreover, unlike POD-DOPE vesicles, they do not show a
concentration-dependent release rate at low pH (Supporting Infor-
mation). This is evidence for a noncontact-mediated content
release.14 When the OEPC liposomes were incubated in the pH
5.2 buffer, the average particle diameter decreased from 154 to
130 nm in 10 min, then stabilized for more than 10 h even at pH
1.0. TLC analysis showed that OEPC was completely hydrolyzed
to the lyso-PC. However, the hydration of a lipid film of lyso-PC/
dodecanol (mole ratio 1:1) only led to micelles instead of liposomes.
Supporting Information Available: Synthetic and characterization
details for all compounds and experimental protocols for liposome
studies. This material is available free of charge via the Internet at
References
(1) Drummond, D. C.; Zignani, M.; Leroux, J. Prog. Lipid Res. 2000, 39,
409-460.
(2) Simoes, S.; Moreira, J. N.; Fonseca, C.; Duzgunes, N.; de Lima, M. C.
AdV. Drug DeliVery ReV. 2004, 56, 947-965.
(3) Guo, X.; Szoka, F. C. Acc. Chem. Res. 2003, 36, 335-341.
(4) Guo, X.; Szoka, F. C. Bioconjugate Chem. 2001, 12, 291-300.
(5) Gerasimov, O. V.; Boomer, J. A.; Qualls, M. M.; Thompson, D. H. AdV.
Drug DeliVery ReV. 1999, 38, 317-338.
(6) Zhu, J.; Munn, R. J.; Nantz, M. H. J. Am. Chem. Soc. 2000, 122, 2645-
2646.
(7) By, K.; Nantz, M. H. Angew. Chem., Int. Ed. 2004, 43, 1117-1120.
(8) Guo, X.; Huang, Z.; Szoka, F. C. Methods Enzymol. 2004, 387, 147-
152.
(9) Li, W.; Huang, Z.; MacKay, J. A.; Grube, S.; Szoka, F. C., Jr. J. Gene
Med. 2005, 7, 67-79.
(10) Masson, C.; Garinot, M.; Mignet, N.; Wetzer, B.; Mailhe, P.; Scherman,
D.; Bessodes, M. J. Controlled Release 2004, 99, 423-434.
(11) Firestone, R. A.; Pisano, J. M.; Bonney, R. J. J. Med. Chem. 1979, 22,
1130-1133.
(12) Monnard, P. A.; Oberholzer, T.; Luisi, P. Biochim. Biophys. Acta 1997,
1329, 39-50.
(13) Guo, X.; MacKay, J. A.; Szoka, F. C. Biophys. J. 2003, 84, 1784-1795.
(14) Ellens, H.; Bentz, J.; Szoka, F. C. Biochemistry 1985, 24, 3099-3106.
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