Novel amphiphilic probes for [18F]-radiolabeling preformed liposomes and determination of liposomal trafficking by positron emission tomography

Article abstract of DOI:10.1021/jm7010518

Positron-emission tomography (PET) is a noninvasive real-time functional imaging system and is expected to be useful for the development of new drug candidates in clinical trials. For its application with preformulated liposomes, we devised an optimized [

Full text of DOI:10.1021/jm7010518

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J. Med. Chem. 2007, 50, 6454–6457
the tissues in which it becomes concentrated, and its eventual
Novel Amphiphilic Probes for
elimination can be monitored far more quickly and cost
effectively than by the older invasive techniques of killing and
dissecting the animals to obtain similar information.8 In this
study, we applied this idea to DDS drugs.
[18F]-Radiolabeling Preformed Liposomes
and Determination of Liposomal Trafficking
by Positron Emission Tomography
We previously reported the methodology for detecting
noninvasive liposomal trafficking by PET.9,10 In those studies,
we used a water-soluble compound, [2-18F]2-deoxy-2-fluoro-
glucose ([18F]FDG), encapsulated inside the vesicles. To
encapsulate the [18F]FDG, we restructured the lipid bilayer by
repeating freeze–thaw cycles. This method was not sophisticated
in terms of efficiency of radiolabeling and prevention of
occupational irradiation. Recently, Marik et al.11 synthesized a
radiolabeled amphiphilic compound for determining liposomal
distribution by PET, although their method is not applicable to
preformed liposomes.
Takeo Urakami,† Shuji Akai,‡ Yurie Katayama,†
Norihiro Harada,§ Hideo Tsukada,§ and Naoto Oku*,†
Department of Medical Biochemistry and Global COE and
Department of Synthetic Organic Chemistry, Graduate School of
Pharmaceutical Sciences, UniVersity of Shizuoka, Yada, Suruga-ku,
Shizuoka 422-8526, Japan, and PET Center, Central Research
Laboratory, Hamamatsu Photonics K.K., Hirakuchi, Hamakita-ku,
Hamamatsu 434-8601, Japan
ReceiVed August 24, 2007
In the present study, we developed not only novel [18F]-
positron-labeled compounds for liposomal labeling but also a
new universal methodology for rapid and one-step labeling of
preformed liposomes. Potent compounds were selected from a
diversity of synthesized amphiphilic compounds through a series
of in vitro nonradioisotope (non-RI) and RI screening studies.
On the basis of the results of these studies, the structures of
[18F]-labeled compounds were optimized. By use of these
compounds, liposomes were [18F]-labeled with high efficiency
and liposomal trafficking in mice was visualized by real-time
analysis using a planar positron imaging system (PPIS).
To conduct the experiments for a preliminary screening of
liposome-labeling compounds (vide infra), we designed novel
amphiphilic compounds (2aA-2cE) and prepared them as
follows: Sharma’s Yb(OTf)3-catalyzed etherification method12
was applied to the coupling of the known benzyl alcohol 1a13
having a lipophilic n-octyl group to commercial diethylene
glycol. The reaction proceeded gradually at 50 °C to afford 2aA.
Commercial poly(ethylene glycol)s (PEGs) with average mo-
lecular weights of 200, 285–315, 380–420, and 850–950 were
used for preparation of 2aB, 2aC, 2aD, and 2aE, respectively.
In these cases, the reactions proceeded at ambient temperature.
Whereas 2aB was obtained as a single compound having four
PEG units after purification via SiO2 chromatography, others
were mixtures of several analogues having different lengths of
the PEG chain, and the average number of its PEG units was
indicated by “m”. Similarly, 2bA-2cE were synthesized by the
reaction of 1a,b13 with diethylene glycol or the corresponding
PEG.
The toluene sulfonates (3bA-3bC and 3cA) were obtained
by reactions of the sodium alkoxides derived from the corre-
sponding 2 with TsCl.14 Purification of crude products via SiO2
chromatography afforded 3bA-3bC and 3cA, each as a single
compound. In the same manner, the toluene sulfonates (6a and
6b) without the aromatic group were prepared from com-
mercially available 5a,b. The fluorinated compounds
(4bA-4bC, 4cA, 7a, and 7b) were prepared as references for
the [18F]-labeled compounds ([18F]4 and [18F]7) by the direct
fluorination of the corresponding alcohols (2 and 5) with
(diethylamino)sulfur trifluoride (DAST, Scheme 1).15 The
preparation of the [18F]-labeled compounds ([18F]4bA-4bC,
[18F]4cA, [18F]7a, and [18F]7b) was effectively achieved via
nucleophilic substitution of the corresponding toluene sulfonates
(3 and 6) with [18F]KF/K[2.2.2] obtained by the previously
reported method16 with minor modifications (Scheme 2).
Abstract: Positron-emission tomography (PET) is a noninvasive real-
time functional imaging system and is expected to be useful for the
development of new drug candidates in clinical trials. For its application
with preformulated liposomes, we devised an optimized [18F]-compound
and developed a direct liposome modification method that we termed
the “solid-phase transition method”. We were successful in using
1-[18F]fluoro-3,6-dioxatetracosane ([18F]7a) for in vivo trafficking of
liposomes. This method might be a useful tool in preclinical and clinical
studies of lipidic particle-related drugs.
Liposomes can be used as multipurpose drug carriers in a
wide range of applications. Liposomes have been used as one
of the most ideal drug carriers for anticancer agents, antifungal
antibiotics, photosensitizers, nucleic acid derivatives for gene
therapy, and so on.1–4 From the viewpoint of drug delivery
systems (DDSsa) the pharmacokinetics, pharmacodynamics, and
pharmacotoxics of such drugs have been improved through the
formulation of liposomes or other kinds of lipidic particles such
as lipid complexes and microspheres. To use liposomes as drug
carriers, it is necessary to consider many properties of liposomal
formulations, e.g., lipid composition, vesicular size, surface
electrostatic potential, and functional modification, that influence
independently or mutually the pharmacological characteristics
of the liposomes.5 Comprehensive evaluation of liposomes in
the living body is therefore important for the development of
liposomal DDS drugs and in DDS studies. For evaluation of
the pharmacokinetics of liposomes in vivo, noninvasive real-
time imaging of liposomes is one of the ideal techniques.
Positron emission tomography is a noninvasive technique and
has been used for the clinical functional diagnosis in many
applications related to oncology, neurology, cardiology, psy-
chiatry, and so on.6,7 Also, this technique can be applied in
preclinical studies. Once a positron-labeled candidate drug is
injected into animals, the distribution of the drug in the body,
* To whom correspondence should be addressed. Phone: +81-54-264-
5701. Fax: +81-54-264-5705. E-mail: oku@u-shizuoka-ken.ac.jp.
†
Department of Medical Biochemistry and Global COE, University of
Shizuoka.
‡
Department of Synthetic Organic Chemistry, University of Shizuoka.
Hamamatsu Photonics K.K.
§
aAbbreviations: PET, positron emission tomography; DDS, drug delivery
systems; FDG, 2-deoxy-2-fluoroglucose; RI, radioisotope; PEG, poly(eth-
ylene glycol); DAST, (diethylamino)sulfur trifluoride; HPLC, high-
performance liquid chromatography; FBS, fetal bovine serum; HDL, high-
density lipoprotein; DMSO, dimethyl sulfoxide; PBS, phosphate buffered
saline; PPIS, planar positron imaging system.
10.1021/jm7010518 CCC: $37.00
2007 American Chemical Society
Published on Web 12/01/2007

Letters
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6455
Scheme 1a
Figure 1. Efficiency of incorporation of novel amphiphilic compounds
into liposomes. Incorporation experiments were performed by the solid-
phase transition method, and the amount of compound in each fraction
(liposome, supernatant, and residue) was determined by HPLC.
Figure 2. Stability of modified liposomes. Liposomes modified with
nonradiolabeled amphiphilic compounds were incubated for 1 h in the
presence of FBS, and the amounts of compounds in the liposome were
determined. Data represent the mean ( standard deviation (n ) 4).
a Reagents and conditions: (a) H(OCH2CH2)mOH, Yb(OTf)3, ClCH2-
CH2Cl, 50 °C (for 2A), room temp (for 2B–E), 10–50% yields; (b) NaH,
THF, 0 °C to room temp, then TsCl, room temp, 40–70% yields; (c) DAST,
CH2Cl2, 0 °C to room temp, 10–35% yields.
analysis of particle size in water (data not shown) led us to
consider that these compounds had formed micelles.
On the basis of the above-mentioned experiments, we selected
four compounds (2bA–2bC, and 2cA) that had relatively high
incorporation efficiency and different physicochemical properties
and examined the stability of incorporation of these compounds
in the presence of serum. Liposomes were incubated at 37 °C
for 1 h in the presence of 50% fetal bovine serum (FBS). It is
well-known that amphiphilic compounds associated with the
lipid bilayer of liposomes via weak hydrophobic interactions
are transferred from liposome to serum, and that phenomenon
is due mainly to high-density lipoprotein (HDL) in the serum.
Liposomes and other components including serum lipoproteins
were fractionated by gel filtration chromatography, and the
amount of compounds that remained in the liposomal fraction
was determined. Strikingly, there were significant differences
among the compounds in terms of stability in serum (Figure
2), and a structure–activity relationship was indicated.
Scheme 2a
a Reagents and conditions: (a) [18F]KF/K[2,2,2], MeCN, reflux, 10 min.
Next, the practically useful compounds 1-[18F]fluoro-3,6-
dioxatetracosane ([18F]7a) and (Z)-1-[18F]fluoro-3,6-dioxatet-
racos-15-ene ([18F]7b) were prepared, and the efficiency of
liposomal labeling using these [18F]-labeled compounds was
determined. Since the [18F]-labeled compounds possessed very
high radioactivity, only a very small amount of compound was
needed for liposomal labeling in comparison with the amount
of nonradiolabeled compounds used in the non-RI experiment
mentioned above. Therefore, it is possible to conclude a
difference in labeling efficiency between a trace amount of
radiolabeled compounds and UV detectable non-RI compounds
by use of the “solid-phase transition method”. Figure 3 indicates
the labeling efficiency of [18F]-labeled compounds (4bA-4bC,
4cA, 7a, and 7b). There was no significant difference between
it and that of the corresponding nonradiolabeled compounds.
We then examined the stability of [18F]-labeled compounds.
Figure 4 indicates that there was no significant difference in
In this study, we developed a new method of liposomal
labeling named the “solid-phase transition method” and deter-
mined the incorporation efficiency of the diverse nonradiolabeled
compounds (2aA-2cE). At first, the amphiphilic compounds
(2aA-2cE) were dried to make a thin film. Liposomes were
added to the solvent-free compound (100:1 as the molar ratio
of lipids to compounds) and incubated at 65 °C for 15 min.
Then the liposomal solutions were transferred to centrifuge
tubes, and the compounds not incorporated were removed by
an ultracentrifugation. Compounds in the liposomal fraction
(pellet), supernatant, and residue were quantified by HPLC.
Figure 1 shows the incorporation efficiency of the various
compounds. In the case of some compounds such as 2aD and
2aE with a shorter aliphatic hydrocarbon chain and a longer
PEG chain, the incorporation efficiency was decreased. The

6456 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Letters
Figure 3. Incorporation efficiency of [18F]-radiolabeled amphiphilic
compound. Incorporation experiment with [18F]-radiolabeled probe was
performed by the solid-phase transition method. The incorporation
efficiency was determined by measuring γ-ray radioactivity. The data
represent the mean ( standard deviation (n ) 4).
Figure 5. Whole-body imaging of [18F]7a and liposome labeled with
[
18F]7a in BALB/c mice by use of PPIS. Each radiolabeled sample at
a dose of 2.5 MBq was injected via a tail vein. (A) Data were acquired
with a 1 min frame at 1, 10, 20, 30, 40, 50, and 60 min after injection.
Liposomes with diameters of 170 and 570 nm were accumulated in
spleen (white arrowheads) and liver (green arrowheads). (B) Shown is
the biodistribution in BALB/c mice after intravenous injection of
[
18F]7a or liposomes labeled with [18F]7a.
were also supported by the data from radioactivity at autopsy
(Figure 5B). In contrast, the free compound accumulated in the
kidneys immediately after injection and was then excreted in
the urine. This result reflected the nature of liposomes. These
results suggest that this novel methodology of noninvasive real-
time whole-body imaging enables us to visualize the behavior
of various kinds of liposomes or lipidic particles such as lipid
complexes and lipid microspheres. Also, this technique may be
applied to larger animal models and three-dimensional imaging
provided by PET. In the future, it may be possible to use this
technique for the detection of liposomal behavior in preclinical
and clinical studies. Moreover, when lipidic particles specifically
targeted to disease sites are developed, these particles will be
useful not only for drug delivery but also for diagnostic imaging
of the sites by use of the present technology.
In summary, here we introduced novel [18F]-labeled am-
phiphilic compounds for liposomal modification that were highly
incorporated into liposomes and stable in serum. This universal
method of liposomal modification, i.e., the solid-phase transition
method, can be used for various kinds of liposomes and lipidic
particles. The compounds are useful as PET probes for imaging
liposomal trafficking in the living body. An in vivo study with
PPIS revealed the feasibility of the developed 18F-probe. These
findings suggest that [18F]7a is a promising PET probe for
imaging of liposomal behavior. Furthermore, only one or a few
kinds of 18F-probe can be applied to various kinds of liposomes
and lipidic particles. Further study should clarify the useful-
ness and utility of the current probes and methodology in
preclinical and clinical studies on lipidic particle-based DDS
drugs.
Figure 4. Stability in the serum of liposomes modified with [18F]-
radiolabeled amphiphilic compounds. Liposomes modified with [18F]-
radiolabeled amphiphilic compounds were incubated in the presence
or absence of FBS for 30 min. The reactants were fractionated by gel
filtration chromatography, and the radioactivity of each fraction was
measured. (A) Typical elution patterns are shown: liposome fractions
5–9; serum lipoprotein fractions 9–16. (B) The radioactivity in liposome
fractions is shown.
stability in liposomes between non-RI and RI compounds. These
results proved the feasibility of screening with nonradiolabeled
compounds to obtain appropriate radiolabeled compounds.
Interestingly enough, although there is only one difference in
the structures between [18F]7a and [18F]7b, which is the presence
of an unsaturated bond in the aliphatic hydrocarbon chain of
the latter, the stability of [18F]7a in serum was much better.
Therefore, we selected [18F]7a as the probe with the most
potential for in vivo imaging of liposomes.
It is well-known that one of the most important elements in
trafficking of liposomes in vivo is the diameter of the particles.
Larger-sized liposomes are quickly trapped by the reticuloen-
dothelial system (RES) in the liver and spleen. To demonstrate
the usefulness of [18F]7a, we examined the typical in vivo
behavior of differently sized liposomes. We prepared 18F-labeled
liposomes with three different diameters, namely, 90, 170, and
570 nm, as well as the liposome-free compound dispersed in
1% DMSO/PBS, and injected them into mice via a tail vein.
Whole-body imaging of [18F]7a in normal mice was obtained
by using PPIS. Real-time imaging from PPIS showed that the
larger the liposomes, the more the liposomes were trapped and
accumulated in the spleen (Figure 5A). These imaging results

Letters
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6457
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Acknowledgment. We acknowledge the technical assistance
of Dr. T. Kakiuchi in the in vivo study (Hamamatsu Photonics
K.K.) and the chemical synthesis conducted by T. Baba, N.
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by a grant from Central Shizuoka Cooperation of Innovative
Technology and Advanced Research in Evolution Area (City
Area) supported by the Ministry of Education, Culture, Sports,
Science, and Technology, Japan.
Supporting Information Available: Experimental procedures
and characterization data for all new compounds; details of
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and RI compounds, and mice positron imaging studies. This material
is available free of charge via the Internet at http://pubs.acs.org.
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