Guanidine-containing molecular transporters: Sorbitol-based transporters show high intracellular selectivity toward mitochondria

Article abstract of DOI:10.1002/anie.200701346

(Figure Presented) Special delivery: Transporters constructed on a sorbitol scaffold with eight guanidine residues show significant translocation across the cell membrane and the blood-brain barrier and high affinities toward mitochondria in HeLa and KG1a

Full text of DOI:10.1002/anie.200701346

Communications
DOI: 10.1002/anie.200701346
Drug Delivery
Guanidine-Containing Molecular Transporters: Sorbitol-Based
Transporters Show High Intracellular Selectivity toward
Mitochondria**
Kaustabh K. Maiti, Woo Sirl Lee, Toshihide Takeuchi, Catherine Watkins, Marjan Fretz,
Dong-Chan Kim, Shiroh Futaki, Arwyn Jones, Kyong-Tai Kim, and Sung-Kee Chung*
The therapeutic efficacy of a drug depends on its ability to
reach the desired target tissues, cells, and even intracellular
organelles. However, these biological structures are highly
protected by various membranes that have evolved in such a
way that only a very limited number of foreign molecules are
allowed to enter them. The biological barriers include, for
example, cellular plasma membranes, the blood–brain barrier
(BBB), and nuclear and mitochondrial membranes. It is
generally known that the plasma membrane allows entrance
only to those molecules with an appropriate range of
molecular size, polarity, and charge. Hence, many drug
candidates with promising in vitro activities fail to be
developed into useful pharmaceutical agents.
pedia protein of Drosophila, and related peptides[1–4] have
been extensively studied to improve the absorption, distribu-
tion, metabolism, and elimination (ADME) properties of
poorly bioavailable drugs including small molecules,[5] pro-
teins,[6,7] nucleic acids, and genes.[8,9] However, the peptide-
based molecular transporters are susceptible to a variety of
endogenous proteases in the body, thus limiting their
bioavailability.
More recently, several research groups have developed
synthetic molecular transporters based on peptoids,[10] oligo-
carbamates,[11] b-peptides,[12,13] peptide nucleic acids,[14] and
others.[15–17] We also have recently reported a novel class of
guanidine-containing molecular transporters based on inosi-
tol dimers as the scaffold. These structures were synthesized
by connecting two units of myo- or scyllo-inositol through a
carbonate or amide linkage, and multiple units of the
guanidine functionality were elaborated onto these scaffolds
by peracylation with an w-aminocarboxylate derivative of
varying chain lengths.
The production of molecular transporters to overcome
these biological barriers would be highly desirable in drug
development. In this context, a number of cell-penetrating
peptides (CPPs) derived from HIV-1 Tat protein, Antenna-
[*] Dr. K. K. Maiti, W. S. Lee, Prof. Dr. S.-K. Chung
Department of Chemistry
These transporters exhibited very high membrane-trans-
locating properties in simian kidney (COS-7), mouse macro-
phage (RAW264.7), and HeLa cells, and they were distrib-
uted largely in cytosol. Their internalization mechanism,
although unclear, and localization patterns appeared to be
quite different from those observed with arginine-rich trans-
porters. Furthermore, in the in vivo mouse model studies
some of them were found to cross the BBB efficiently and
show preferential distributions in heart, lung, and brain over
liver, kidney, and spleen.[18,19] These results have strongly
suggested to us that achieving intracellular and/or tissue
selectivity might be possible by varying the structural
components in the molecular transporter design, such as
scaffold, linker chain, and number of guanidine residues, with
concomitant modifications of physicochemical properties.
Herein, we report molecular transporters that exhibit high
selectivity for mitochondrial localization and also overcome
the BBB.
Pohang University of Science and Technology
Pohang 790-784 (Korea)
Fax: (+82)54-279-3399
E-mail: skchung@postech.ac.kr
Homepage: http://www.postech.ac.kr/chem/skc~lab
T. Takeuchi, Prof. Dr. S. Futaki
Institute for Chemical Research
Kyoto University
Uji, Kyoto 611-0011 (Japan)
and
SORST, Japan Science and Technology Corporation (JST)
Kawaguchi, Saitama 332-0012 (Japan)
D.-C. Kim, Prof. Dr. K.-T. Kim
Department of Life Science
Pohang University of Science and Technology
Pohang 790-784 (Korea)
C. Watkins, M. Fretz, Prof. Dr. A. Jones
Welsh School of Pharmacy
Cardiff University
Cardiff CF103XF (UK)
Although the previously reported G8 molecular trans-
porters based on dimeric inositol scaffolds showed some
interesting and useful properties, such as BBB penetration,
we decided to change the scaffold and the linking pattern
while staying with eight units of the guanidine residue. Thus,
we have designed a series of molecular transporters based on
the sorbitol scaffold and bifurcated linkers each carrying two
residues of guanidine. Sorbitol (d-glucitol) occurs widely in
plants, especially in the Rosaceae family, which includes
apples, pears, cherries, and apricots. Sorbitol is commercially
[**] We thank Sun-Hee Lee for technical assistance involving cell culture
and CLSM operation at Postech. Financial support from BK-21 and
MOST (National Frontier Research Program administered through
KRICT/CBM), and Grants-in-Aid for Scientific Research from the
Ministry of Education, Culture, Sports, Science, and Technology of
Japan are gratefully acknowledged. T.T. is grateful for a JSPS
research fellowship for young scientists. Part of this work (A.J.,
C.W.) was supported by BBSRC.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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produced by reducing d-glucose or d-glucono-1,4-lactone,
and is extensively used in the food industry, as it is highly
water soluble and free of any discernible toxicity.[20]
6-O-Trityl-a-d-glucose, prepared from a-d-glucose
according to the literature procedure,[21,22] was reduced with
NaBH4 in methanol to produce 6-O-trityl-d-sorbitol. The
primary hydroxy group in the tritylated sorbitol was selec-
tively protected with tert-butyldiphenylsilyl chloride
(TBDPS-Cl) and triethylamine (TEA) in pyridine to afford
1-O-(tert-butyldiphenylsilyl)-6-O-trityl-d-sorbitol (1, see
Scheme 1) in good yield (see Scheme 1 in the Supporting
Information for details).
Two types of branched chain containing the bisguanidine
moiety were prepared. In the first type, a double Michael
addition of w-aminocarboxylate (with varying chain length,
n = 5 and 7) to a large excess of acrylonitrile in glacial acetic
acid afforded the dicyanoethylated w-aminocarboxylate
products in good yields.[23,24] The cyano functionality was
quantitatively converted to the corresponding amino group
by catalytic hydrogenation over Raney Ni in 1n NaOH. The
guanidinylation of these intermediates with N,N’-di-Boc-N’’-
trifluoromethanesulfonylguanidine and TEA in 1,4-dioxane/
water (5:1) provided the requisite bisguanidinylated carbox-
ylic acids 2a and 2b (see Scheme 1) in reasonable yields (68
and 74%).
For construction of the second type of branched chain, the
polyamidoamine dendrimer-type process was employed.
Thus, the Michael addition between methyl acrylate and 6-
aminocaproic acid, followed by exhaustive amidation of the
resulting diester with an excess amount of ethylenediamine,
gave the bisaminocarboxylate after thorough removal of
ethylenediamine by co-evaporation with toluene. The guani-
dinylation of the amino groups as previously described gave
the bisguanidinylated carboxylic acid 3 (see Scheme 2) in
68% yield after column chromatography (see Scheme 2 of the
Supporting Information for details).
The exhaustive acylation of the scaffold, 1-O-(tert-butyl-
diphenylsilyl)-6-O-trityl-d-sorbitol (1), with bisguanidiny-
lated carboxylic acids 2a and 2b in dry CH2Cl2 in the
presence of EDC and DMAP gave compounds 4a and 4b in
good yields after column chromatography on silica gel
(Scheme 1). Selective removal of the TBDPS group in 4
with TBAF in dry THF, to give 5, was followed by the
introduction of a new amino functionality on that hydroxy
group. Thus, acylation of 5 with Cbz-protected aminocaproic
acid under the EDC coupling conditions furnished the desired
products 6 after the usual workup and purification. After
deprotection of the N-Cbz group in 6 to give 7, a fluorescence
probe was attached by reaction with fluorescein-5-isothio-
cyanate (FITC) to afford the FITC-labeled compounds 8 in
approximately 70% yield after purification by flash column
chromatography. The target molecular transporters 9a and 9b
were obtained as HCl salts by deprotecting the Boc groups on
the guanidine moieties with ethyl acetate saturated with
gaseous HCl, and purification by medium-pressure liquid
chromatography (MPLC) on a reversed-phase (C8) column
(Scheme 1).
Scheme 1. Synthesis of transporters 9; a: n=5; b: n=7. Reagents and
conditions: a) 2, EDC, DMAP, CH2Cl2, RT, 30 h (4a, 66%; 4b, 92%);
b) TBAF, THF, RT, 10 h (5a, 79%; 5b, 68%); c) N-Cbz-protected
aminocaproic acid, EDC, DMAP, CH2Cl2, RT, 10 h (6a, 71%; 6b,
74%); d) 10% Pd/C (10 mol%), H2 (1 atm), MeOH, RT, 25 h (7a,
91%; 7b, 87%); e) FITC (isomer I), TEA, absolute EtOH/THF (5:2),
RT, dark, 24 h (8a, 68%; 8b, 61%); f) HCl(g), EtOAc, RT, 20 h (9a,
81%; 9b, 86%). EDC=3-(3-dimethylaminopropyl)-1-ethylcarbodii-
mide; DMAP=4-(dimethylamino)pyridine; TBAF=tetrabutylammo-
nium fluoride; Cbz=benzyloxycarbonyl; Boc=tert-butoxycarbonyl.
conditions to obtain 10 (Scheme 2), which was then trans-
formed to another type of molecular transporter 15 by
procedures analogous to those described for compounds 9.
All key intermediates and the final target compounds 9a, 9b,
and 15 were thoroughly characterized by HPLC, NMR
spectroscopy, and MALDI-TOF mass spectrometry.
Preliminary evaluations of the synthetic transporters for
their uptake ability in HeLa cells were carried out by confocal
laser scanning microscopy (CLSM). Even after incubation for
15 min at 378C with 10 mm of transporters, cells were
substantially marked by fluorescence. The cells were analyzed
without fixing and the extents of cellular uptake were visually
compared with FITC-labeled arginine nonamer (R9-Fl) as the
reference. A significant fluorescence signal was observed for
9a, 9b, and R9-Fl, but internalization of 15 appeared to be
much less than with the others.
Next, we introduced the alternative linker 3 into the
sorbitol scaffold 1 by exhaustive acylation under the EDC
More detailed fluorescence-activated cell-sorter (FACS)
studies were carried out in terms of the concentration and
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Figure 1. Cellular uptake of sorbitol-based transporters. A) FACS quan-
tification of the transporters taken up by HeLa cells. Cells were treated
with transporters 9a, 9b, 15, and FITC-labeled R8 peptide in serum-
containing medium at 378C for 1 h and then analyzed by FACS. Open
and filled columns represent the uptake by the cells treated with 10
and 20 mm transporters, respectively. B) Kinetics in cellular uptake of
~
&
transporters. HeLa cells were incubated with 9a ( ), 9b ( ), and 15
^
( ) (10 mm each) at 378C for the time indicated and analyzed by
FACS. Data shown means with Æ standard deviation from three
samples.
Scheme 2. Synthesis of transporter 15. Reagents and conditions: a) 3,
EDC, DMAP, CH2Cl2, RT, 30 h, 62%; b) TBAF, THF, RT, 15 h, 80%;
c) N-Cbz-protected aminocaproic acid, EDC, DMAP, CH2Cl2, RT, 18 h,
72%; d) 10% Pd/C (10 mol%), H2 (1 atm), MeOH, RT, 20 h, 87%;
e) FITC (isomer I) (1.2 equiv), TEA, absolute EtOH/THF (5:2), RT,
dark, 24 h, 65%; f) HCl(g), EtOAc, RT, 24 h, 83%.
time dependency (Figure 1A and B). The R8 peptide is
known as a representative peptide among the transporters
that show highly efficient internalization.[2] Analyzed by the
relative fluorescence intensities, 9a and 9b (at 10 mm) had an
efficiency almost comparable to that of FITC-labeled R8
(FITC-R8; 10 mm; Figure 1A). On the other hand, the
internalization efficiency of 15 was not nearly as high. In
addition, a substantial increase of cellular uptake of 9a and 9b
was observed when the concentration was raised to 20 mm.
Figure 1B shows the kinetics of cellular uptake of these
transporters. The cellular uptake almost reaches a plateau in
approximately 3 h with 9b again showing the highest effi-
ciency.
Confocal microscopic observation of the cells treated with
these transporters demonstrated that 9a and 9b yielded very
similar cytoplasmic distributions (Figure 2A). Transporter 15
gave only very faint fluorescence signals in the same experi-
ment. As it seemed likely that 9a and 9b co-localized with
some vesicular components with very similar cytoplasmic
localization, we selected 9a for a more detailed study of
cytoplasmic localization of the vectors. Internalization of 9a
Figure 2. Cellular localization of sorbitol-based transporters. A) Confo-
cal microscopic observation of HeLa cells treated with 9a, 9b, and 15
(10 mm each) at 378C for 1 h. B) Mitochondrial localization of trans-
porter 9a. The cells were treated with 9a (green; 10 mm) and
MitoTracker (red; 250 nm) at 378C for 1 h. No significant co-local-
ization of the signals of 9a (green) with C) Tat peptide (10 mm),
D) transferrin (25 mgmLÀ1) labeled with tetramethylrhodamine, and
E) LysoTracker (50 nm, red) was observed after co-incubation at 378C
for 1 h. F) A significant decrease in internalization of 9a (10 mm)
occurred on incubation at 48C for 1 h. Scale bars: 20 mm.
was significantly inhibited at 48C (Figure 2F), apparently
suggesting an involvement of energy-dependent steps in this
internalization. Figure 2C–E clearly shows that 1) there is
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little or no co-localization between 9a and tetramethylrhod-
amine-labeled Tat peptide or transferrin, which strongly
suggests a different uptake mechanism for 9a from those of
Tat peptide and transferrin, and 2) a major portion of 9a did
not reach lysosomes or acidic endosomes stained by Lyso-
Tracker in 1 h. However, to our surprise significant co-
localization of 9a and mitochondria-specific dye, MitoTracker
Red, was observed (Figure 2B and Figure 1 of the Supporting
Information), which suggests accumulation of 9a in mito-
chondria after the cellular uptake. Transporter 9b also
showed similar co-localization with MitoTracker in HeLa
cells (data not shown). These observations are highly
significant, especially as Tat and the related peptides do not
show significant mitochondrial localization.[25]
combined with p-electron density delocalization over more
than three atoms (“delocalized cations”).[26] As the mitochon-
drial membrane is known to have a high membrane potential
with negative charges present on the cytosolic side, it can be
readily imagined that the positive charges present on the
guanidinium residues of the transporter help its interaction
with the mitochondrial membrane. However, it is not clear at
this stage what specific structural features (for example,
sorbitol scaffold, number of guanidine units, or branched-
linker pattern) of the transporters are responsible for the
observed organellar selectivity, especially as neither R8 nor
the dimeric inositol-based transporters show this selectiv-
ity.[18,25] However, in two very different cell lines there is clear
sequestration of these compounds in mitochondria, which
suggests that they may be useful vectors for mitochondrial
delivery.
Molecular transporters capable of selectively delivering a
drug or cargo to mitochondria are desperately needed in the
diagnosis and treatment of mitochondrial diseases.[26]
A
Next, we examined the tissue-distribution patterns of
compound 9a in mice (Figure 3). Transporter 9a (HCl salt,
82 mgkgÀ1) was dissolved in sterile distilled water and
heterocyclic tetraguanidinium compound, which has a skel-
etal frame somewhat reminiscent of those of the heterocya-
nine dyes and the cyclic iminium structure of MitoTracker
Red, was reported to target mitochondria,[16] but its potential
utility might be somewhat limited because the observation
was made with fixed cells and it showed significant toxicity
even at 10 mm. The novel structure and synthetic ease of
transporters 9a and 9b may provide a new platform for the
design of further mitochondrial-targeting systems.[27]
Mitochondrial defects are known to occur in a wide
variety of degenerative diseases, aging, and even cancer.
Abnormal mitochondria have been associated with many
diseases including maternally inherited Leber hereditary
optic neuropathy (LHON), progressive external ophthalmo-
plegia (PEO), Kearns–Sayre Syndrome (KSS), mitochondrial
encephalomyopathy, lactic acidosis and strokelike syndrome
(MELAS), myoclonic epilepsy and ragged-red fibers
(MERRF), and a cluster of metabolic diseases (syndro-
me X).[28] In addition, mitochondria are known to play key
roles in apoptosis (cancer therapy),[29] familial amyotrophic
lateral sclerosis (ALS, Lou Gehrigꢀs disease),[30] Huntingtonꢀs
disease,[31] and Alzheimerꢀs disease.[32]
In view of the significant observation that these trans-
porters efficiently migrate to mitochondria in HeLa cells, the
cellular distribution of compound 9b was also analyzed in
CD34+ stem-cell-like KG1a leukemia cells, which had pre-
viously been characterized with respect to interactions with
CPPs.[33,34] KG1a cells incubated with compound 9b for 1 h at
378C are clearly labeled but there is some heterogeneity with
respect to the degree of labeling (see Figure 2 of the
Supporting Information).[35] Co-incubation experiments with
9b and MitoTracker confirmed our previous analyses in HeLa
cells, again showing extensive co-localization. The full extent
of co-localization is most apparent when the cells are
analyzed through the xyz axes.
Figure 3. Distribution of 9a (HCl salt) in mouse tissue (bottom; top:
control). Fluorescence micrographs of heart muscle (a, b), spleen
(c, d), liver (e, f), kidney (g, h), and lung (i, j) tissue sections, and of
coronal brain sections (k, l) isolated from mice 20 min after i.p.
injection. Exposure time [ms]: 14000 (a, b); 500 (c, d); 300 (e, f); 500
(g, h); 1200 (i, j); 15000 (k, l); lmax =488 nm (green fluorescence from
FITC).
injected intraperitoneally (i.p.) into 8-week-old mice
(C57BL/6). After 20 min, the treated mice were perfused
with 4% paraformaldehyde in phosphate buffer solution
(PBS; pH 7.4), and the organs including the heart, spleen,
liver, kidneys, lungs, and brain were incubated overnight in a
solution of 0.5m sucrose in PBS. Placed in cryoprotectant,
they were cut into 15-mm sections with a cryostat and
transferred to coated glass slides. After drying at 378C, the
sections were washed with PBS and treated with 0.3% Triton
X-100 for 15 min at room temperature, and analyzed with an
Axioplan2 fluorescence imaging microscope (Carl Zeiss).
The Tat peptides were previously reported to show wide
distributions in the liver, kidneys, lungs, heart muscle, and
spleen,[36,37] and the synthetic transporters based on the
dimeric inositol scaffold were shown to be distributed
predominantly in the heart, lung, and brain tissues.[18] With
transporter 9a, a higher distribution was found in the heart
muscle and brain sections than in any other tissue examined.
It might be possible that the observed tissue selectivity is
related to the organellar selectivity of 9a toward mitochon-
dria, as the heart and brain are presumably quite active in
energy metabolism. An alternative possibility might be that
Although mitochondrial localization sequences (MLSs)
and endogenous mitochondrial metabolite transporters have
been reported, it is unlikely on a structural basis that the
selective targeting of compounds 9a and 9b is related to
these. Rhodamine 123 and cyanine dyes are the best-known
mitochondriotropic compounds, and the structural features
required for affinity have been proposed to be amphiphilicity
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[12] N. Umezawa, M. A. Gelman, M. C. Haigis, R. T. Raines, S. H.
Gellman, J. Am. Chem. Soc. 2002, 124, 368.
[13] N. Rueping, Y. Mahajan, M. Sauer, D. Seebach, ChemBioChem
2002, 3, 257.
[14] P. Zhou, M. Wang, L. Du, G. W. Fisher, A. Waggoner, D. H. Ly, J.
Am. Chem. Soc. 2003, 125, 6878.
[15] N. W. Luedtke, P. Carmichael, Y. Tor, J. Am. Chem. Soc. 2003,
125, 12374.
[16] J. Fernandez-Carneado, M. Van Gool, V. Martos, S. Castel, P.
Prados, J. de Mendoza, E. Giralt, J. Am. Chem. Soc. 2005, 127,
869.
[17] H. H. Chung, G. Harms, C. M. Seong, B. H. Choi, C. Min, J. P.
Taulane, M. Goodman, Biopolymers 2004, 76, 83.
[18] K. K. Maiti, O. Y. Jeon, W. S. Lee, D. C. Kim, K. T. Kim, T.
Takeuchi, S. Futaki, S. K. Chung, Angew. Chem. 2006, 118, 2973;
Angew. Chem. Int. Ed. 2006, 45, 2907.
[19] K. K. Maiti, O. Y. Jeon, W. S. Lee, S. K. Chung, Chem. Eur. J.
2007, 13, 762.
[20] J. S. Brimacombe, J. M. Webber in Carbohydrates: Chemistry
and Biochemistry, Vol. 1, 2nd ed. (Ed.: W. Pigman, D. Horton),
Academic Press, New York, 1972, p. 479.
[21] S. K. Chaudhary, O. Hernandez, Tetrahedron Lett. 1979, 20, 95.
[22] A. Klemer, K. Homberg, Chem. Ber. 1961, 94, 491.
[23] F. M. Braæa, G. Dominguez, B. Saez, C. Romerdahl, S. Rob-
inson, T. Barlozzari, Eur. J. Med. Chem. 2002, 37, 541.
[24] M. Dubber, K. T. Lindhorst, Org. Lett. 2001, 3, 4019.
[25] a) M. F. Ross, A. Filipovska, R. A. J. Smith, M. J. Gate, M. P.
Murphy, Biochem. J. 2004, 383, 457; b) M. P. Murphy, R. A.
Smith, Annu. Rev. Pharmacol. Toxicol. 2007, 47, 629.
[26] V. Weissig, S. M. Cheng, G. G. M. DꢀSouza, Mitochondrion 2004,
3, 229.
there is some specific interaction between the transporter and
a cell-surface component of brain and heart tissues.
In summary, in the hope of achieving some intracellular
organellar and/or in vivo tissue selectivity, we have designed
and synthesized transporter molecules based on the sorbitol
scaffold, which is structurally elaborated to carry eight
residues of guanidine base through two different types of
branched chains. One type (9a and 9b) of these transporters
is shown to be efficiently cell-penetrating, and to possess
unique intracellular selectivity toward mitochondria as well as
some interesting tissue selectivity for heart and brain in mice.
At the moment, it is not clear which structural features or
physicochemical properties of these transporters contribute
to the observed selectivity. We speculate that the mitochon-
drial affinity and the preferential distribution in heart and
brain might be related, with potential implications in the
practical delivery of drugs in the therapy of cancers and
diseases related to the central nervous system. Further studies
aimed at elucidating the structure–selectivity relationship of
these guanidine-containing transporters and exploring their
applications are ongoing.
Received: March 28, 2007
Published online: July 2, 2007
Keywords: bioorganic chemistry · carbohydrates · drug delivery ·
.
membranes · mitochondria
[27] The parent structures of the present carriers showed 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
toxicity profiles similar to that of R8, that is, virtually no toxicity
at 10 mm.
[1] Handbook of Cell-Penetrating Peptides, 2nd ed. (Ed.: Ü. Langel),
CRC, Boca Raton, 2006.
[2] S. Futaki, Adv. Drug Delivery Rev. 2005, 57, 547.
[3] S. Futaki, T. Suzuki, W. Ohashi, T. Yagami, S. Tanaka, K. Ueda,
Y. Sugiura, J. Biol. Chem. 2001, 276, 5836.
[28] D. C. Wallace, Science 1999, 283, 1482.
[29] D. R. Green, J. C. Reed, Science 1998, 281, 1309.
[30] V. L. Dawson, Nat. Med. 2004, 10, 905.
[4] A. Joliot, A. Prochiantz, Nat. Cell Biol. 2004, 6, 189.
[5] J. B. Rothbard, S. Garlington, O. Lin, T. Kirschberg, E. Kreider,
P. L. McGrane, P. A. Wender, P. A. Khavari, Nat. Med. 2000, 6,
1253.
[6] J. S. Wadia, R. V. Stan, S. F. Dowdy, Nat. Med. 2004, 10, 310.
[7] I. Nakase, M. Niwa, T. Takeuchi, K. Sonomura, N. Kawabata, Y.
Koike, M. Takehashi, S. Tanaka, K. Ueda, J. C. Simpson, A. T.
Jones, Y. Sugiura, S. Futaki, Mol. Ther. 2004, 10, 1011.
[8] V. P. Torchilin, R. Rammohan, V. Weissig, T. S. Levchenko, Proc.
Natl. Acad. Sci. USA 2001, 98, 8786.
[9] K. Kogure, R. Moriguchi, K. Sasaki, M. Ueno, S. Futaki, H.
Harashima, J. Controlled Release 2004, 98, 317.
[10] P. A. Wender, D. J. Mitchell, K. Pattabiraman, E. T. Pelkey, L.
Steinman, J. B. Rothbard, Proc. Natl. Acad. Sci. USA 2000, 97,
13003.
[31] B. I. Bae, S. Igarashi, M. Fujimoro, N. Agrawal, Y. Taya, S. D.
Hayward, T. H. Moran, C. A. Ross, S. H. Snyder, A. Sawa,
Neuron 2005, 47, 29.
[32] M. Manczak, T. S. Anekonda, E. Henson, B. S. Park, J. Quinn,
P. M. Reddy, Hum. Mol. Genet. 2006, 15, 1437.
[33] S. Al-Taei, N. A. Penning, J. C. Simpson, S. Futaki, T. Takeuchi, I.
Nakase, A. T. Jones, Bioconjugate Chem. 2006, 17, 90.
[34] M. M. Fretz, N. A. Penning, S. Al-Taei, S. Futaki, T. Takeuchi, I.
Nakase, I. G. Storm, A. T. Jones, Biochem. J. 2007, 403, 335.
[35] This kind of heterogeneity has been occasionally observed, and
may be related to cell cycles of individual cells.
[36] S. R. Schwarze, A. Ho, A. Vocero-Akbani, S. F. Dowdy, Science
1999, 285, 1569.
[37] S. R. Cai, G. Xu, M. Backer-Hapak, M. Ma, S. F. Dowdy, H. L.
McLeod, Eur. J. Pharm. Sci. 2006, 27, 311.
[11] P. A. Wender, J. B. Rothbard, T. C. Jessop, E. L. Kreider, B. L.
Wylie, J. Am. Chem. Soc. 2002, 124, 13382.
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