Selective recognition of anionic cell membranes using targeted liposomes coated with zinc(ii)-bis(dipicolylamine) affinity units

Article abstract of DOI:10.1039/c4ob00924j

Zinc(ii)-bis(dipicolylamine) (Zn₂BDPA) coated liposomes are shown to have high recognition selectivity towards vesicle and cell membranes with anionic surfaces. Robust synthetic methods were developed to produce Zn₂BDPA-PEG-lipid conjugates with varying PEG linker chain length. One conjugate (Zn₂BDPA-PEG₂₀₀₀-DSPE) was used in liposome formulations doped with the lipophilic near-infrared fluorophore DiR. Fluorescence cell microscopy studies demonstrated that the multivalent liposomes selectively and efficiently target bacteria in the presence of healthy mammalian cells and cause bacterial cell agglutination. The liposomes also exhibited selective staining of the surfaces of dead or dying human cancer cells that had been treated with a chemotherapeutic agent. the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00924j

Organic &
Biomolecular Chemistry
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Selective recognition of anionic cell membranes
using targeted liposomes coated with
zinc(II)-bis(dipicolylamine) affinity units†
Cite this: Org. Biomol. Chem., 2014,
12, 5645
Serhan Turkyilmaz,a,b Douglas R. Rice,a Rachael Palumboa and Bradley D. Smith*a
Zinc(II)-bis(dipicolylamine) (Zn2BDPA) coated liposomes are shown to have high recognition selectivity
towards vesicle and cell membranes with anionic surfaces. Robust synthetic methods were developed to
produce Zn2BDPA-PEG-lipid conjugates with varying PEG linker chain length. One conjugate (Zn2BDPA-
PEG2000-DSPE) was used in liposome formulations doped with the lipophilic near-infrared fluorophore
DiR. Fluorescence cell microscopy studies demonstrated that the multivalent liposomes selectively and
efficiently target bacteria in the presence of healthy mammalian cells and cause bacterial cell aggluti-
nation. The liposomes also exhibited selective staining of the surfaces of dead or dying human cancer
cells that had been treated with a chemotherapeutic agent.
Received 5th May 2014,
Accepted 18th June 2014
DOI: 10.1039/c4ob00924j
www.rsc.org/obc
Recently, we prepared and evaluated multivalent Zn2BDPA
molecular probes that were equipped with four or more co-
Introduction
Zinc(II)-bis(dipicolylamine) (Zn2BDPA) coordination complexes valently attached Zn2BDPA targeting units and observed high
are known to associate with phosphate polyanions in aqueous selectivity for anionic membranes.29–31 Fluorescent versions of
solution (Fig. 1A) and they have been developed for various these multivalent Zn2BDPA probes were found to be effective
supramolecular applications such as optical chemosensing, optical imaging agents in cell culture and animal models of
biomolecule labeling and catalytic hydrolysis.1–21 We have con- bacterial infection or cell death. A notable finding with the
tributed to this effort by demonstrating that Zn2BDPA com- bacterial studies was the strong propensity of multivalent
plexes have selective affinity for anionic cell membrane Zn2BDPA probes to selectively cross-link and agglutinate bac-
surfaces over the near-neutral membrane surfaces of healthy terial cells. The multivalent Zn2BDPA probes were not bacteri-
mammalian cells.22–28 This discovery has led to molecular cidal, but the agglutination effect was universal regardless of
imaging probes that can target two types of anionic cell Gram-type or cell morphology. These results suggest that
systems with high biomedical significance, namely, bacterial multivalent Zn2BDPA molecular probes have promise as selec-
cells and dead/dying mammalian cells. With bacterial cells, tive, broad spectrum bacterial agglutination agents for infec-
the surrounding envelope is anionic because it contains phos- tion imaging and diagnostics. Similarly, they may also be
phorylated amphiphiles such as phosphatidylglycerol or lipo- incorporated into imaging and theranostic strategies that
techoic acid in the case of Gram-positive bacteria, or lipid A in target mammalian cell death. For example, a hypothetical
the case of Gram-negative bacteria. With dead/dying mamma- cancer treatment strategy might employ a multifunctional
lian cells, the outer membrane surface becomes anionic due drug delivery vehicle to target the sites of endogenous cell
to the exposure of phosphatidylserine during the cell death death within a tumor. If the tumor is responsive to the deli-
process. We have shown that fluorescent Zn2BDPA probes can vered drug, more cell death occurs which, in principle, should
selectively stain these anionic cells even when they are within amplify the drug delivery process during
highly complex and heterogeneous biological environments treatment.32,33
a subsequent
such as cell culture and living animals.22–28
A necessary requirement for each of these potential
imaging and theranostic applications is the construction of
effective multivalent Zn2BDPA imaging probes and drug deliv-
ery vehicles. We were attracted to liposomes as a biocompati-
ble nanoparticle platform whose pharmacokinetic properties
can be optimized by systematic modification of the amphi-
philic building blocks. The ability of non-targeted stealth lipo-
somes to exploit the enhanced permeation retention (EPR)
aDepartment of Chemistry and Biochemistry, 236 Nieuwland Science HallUniversity
of Notre Dame, Notre Dame, IN 46556, USA. E-mail: smith.115@nd.edu
bFaculty of Pharmacy, Department of Pharmaceutical Chemistry, Istanbul University,
34116 Beyazit, Istanbul, Turkey
†Electronic supplementary information (ESI) available: Spectral data, liposome
characterization and cell toxicity data. See DOI: 10.1039/c4ob00924j
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concept of targeted liposomes has alluring features, it also
includes molecular design challenges that must simul-
taneously address conflicting requirements.37–42 Most notable
is the need to coat the liposome surface with polymeric struc-
tures (e.g., PEG chains) that inhibit recognition and premature
clearance of the liposomes by the reticuloendothelial system
(RES). One potential solution is to attach the targeting ligands
to the ends of the polymeric chains, but the current literature
on this strategy does not make it clear if the targeting ligands
will still be available for receptor binding, or if they will trigger
undesired recognition by the RES. There is also the nontrivial
synthetic chemistry challenge of fabricating the targeted lipo-
somes. The most common approach is to append the targeting
ligands to the ends of commercially available polyethylene-
glycol-phospholipid conjugates such as PEG2000-DSPE. There
are two limiting strategies. One is to react the targeting ligand
with preformed liposomes containing a suitably reactive
PEG2000-DSPE. The other approach is to first synthesize the
functionalized PEG2000-DSPE conjugate and then assemble the
liposomes. The latter method provides more opportunity to
validate compositional purity of the liposome components,
but there are still molecule characterization challenges due to
polydispersity of the PEG chains in most commercial supplies
of the starting materials. We believe that studies of targeted
liposomes are best conducted using molecular building blocks
with well-defined chemical structures and high compositional
purity.
Here, we describe the preparation of targeted liposomes
that are coated with multiple Zn2BDPA affinity units through
the incorporation of a small fraction of amphiphilic Zn2BDPA-
PEG-DSPE conjugates in the bilayer membranes (Fig. 1B).
Specifically, we describe the synthesis of two conjugates,
Zn2BDPA-PEG2000-DSPE and Zn2BDPA-PEG500-DSPE (Fig. 1C)
and the in vitro molecular recognition properties of liposomes
that incorporate these conjugates. We find that Zn2BDPA
coated liposomes are able to rapidly cross-link a second popu-
lation of anionic liposomes. Furthermore, we show that
Zn2BDPA coated liposomes can selectively agglutinate bacterial
cells in the presence of healthy mammalian cells and also
selectively target the surfaces of dead/dying mammalian
cancer cells. The results indicate that Zn2BDPA coated lipo-
somes have great promise for wide range of targeted imaging
and drug delivery applications.
Fig. 1 Association of a Zn2BDPA coordination complex with a phos-
phodiester (A), schematic picture of a Zn2BDPA coated liposome with
PEG and Zn2BDPA groups on the inner leaflet omitted for clarity (B),
structures of Zn2BDPA-lipid conjugates used in this study (C).
effect and accumulate inside tumors is well known as one of
the pioneering success stories of nanomedicine.34,35 In related
fashion, stealth liposomes are known to collect in sites of bac-
terial infection within living subjects.36 In both of these cases,
the liposome accumulation process is passive and does not
involve direct molecular targeting. This present report, on the
targeting ability of liposomes that are coated with multiple
Zn2BDPA affinity units, is the first step in a process to develop
targeted liposome systems that enhance the passive accumu-
lation effects described above. There is a large and growing
body of literature on the topic of targeted liposomes; that is,
liposomes coated with targeting ligands having selective
affinity for receptors or biomarkers of disease. Although the
Results and discussion
Synthesis and liposome preparation
Our strategy for the construction of Zn2BDPA-PEG-lipid conju-
gates was largely determined by the commercial availability of
lipids and PEGylated lipids suitable for conjugation. Lipids ter-
minating in amine groups such as DSPE and NH2-PEG2000
-
DSPE are commercially available. Furthermore, bis(dipicolyl-
amine) (BDPA) conjugates of these lipids can be prepared
through standard amide coupling chemistry. The BDPA carb-
oxylic acid derivative 6, which would allow the preparation of
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Scheme 1 Preparation of BDPA carboxylic acid 6. Conditions: (i) LiAlH4, THF, rt, 24 h; (ii) Ethyl 4-bromobutyrate, K2CO3, Bu4NI, 60 °C, 16 h; (iii)
Ph3P, CBr4, DIPEA, THF, rt, 24 h; (iv) 2,2’-dipicolylamine, K2CO3, DMF, rt, 16 h; (v) 2 : 1 : 1 THF–H2O–MeOH, NaOH, reflux, 2 h.
these conjugates, was synthesized in a fashion similar to a DSPE is a polydisperse mixture of PEG chains with a mean
BDPA amine derivative we reported earlier.43 Accordingly, degree of polymerization around 45 and this is reflected in the
dimethyl 5-hydroxyisophthalate (1) was reduced using LiAlH4 HRMS spectrum of 7 (ESI). To remove the mass spectral ambi-
to give 3,5-bis(hydroxymethyl)phenol (2) in high yield,44 which guity regarding the identity of the product and to demonstrate
was coupled with ethyl 4-bromobutanoate using K2CO3/Bu4NI the versatility of the synthetic methods we decided to prepare
in anhydrous DMF to give 3 in 31% yield. Numerous attempts BDPA-PEG500-DSPE (10) from commercially available mono-
at improving the yield for this alkylation proved fruitless. The disperse FmocNH-PEG500-propionic acid (8). Reaction of PfpOH
diol 3 was efficiently converted to the dibromo derivative 4 activated 8 with DSPE proved very sluggish in a wide variety of
using modified Appel conditions.45 The ligands for zinc were solvents due to the poor solubility of this phospholipid. This
installed by reacting 4 with dipicolylamine using K2CO3 in problem was overcome by refluxing the reaction. Removal of
anhydrous DMF to give 5 in good yield. Finally saponification the Fmoc group using piperidine and reaction of the resulting
of 5 afforded the desired BDPA carboxylic acid 6 quantitatively H2N-PEG500-DSPE (9) with PfpOH activated 6 afforded the
(Scheme 1).
desired conjugate 10 in good yield (Scheme 3). It was possible
With 6 in hand we turned our attention to the preparation to purify 10 using a relatively simple preparatory TLC pro-
of BDPA-PEG2000-DSPE (7). A multitude of methods can be con- cedure and the spectral data indicated monodispersity.
sidered for the amide bond formation between
6
and
For cuvette experiments, liposomal dispersions containing
H2N-PEG2000-DSPE.46 We found that the EDC/DMAP mediated 2.5 mol% of the Zn2BDPA-PEG-DSPE amphiphiles were pre-
pentafluorophenol (PfpOH) activation of 6 followed by reaction pared using the film hydration/extrusion method.47 Lipid
with H2N-PEG2000-DSPE reliably afforded BDPA conjugate 7 films composed of 7–cholesterol–POPC or 10–cholesterol–
(Scheme 2). Using this method it was possible to prepare tens POPC (2.5 : 30 : 67.5 mol%, 3.32 µmol total lipid) were hydrated
of milligrams of 7 in one run and purification merely con- using HEPES buffer containing Zn(NO3)2. The Zn2+/BDPA
sisted of a relatively simple preparatory TLC procedure. It molar ratio was 10 to ensure rapid and complete formation of
should be noted that commercially available H2N-PEG2000
-
the Zn2BDPA coordination complexes. In the absence of Zn2+,
the dispersions were impossible to extrude, presumably due to
bilayer self-aggregation caused by the lipophilic nature of the
uncomplexed BDPA units. In the presence of Zn2+, the lipid
films were readily dispersed into aqueous solution and easy to
extrude through polycarbonate membranes with 200 nm dia-
meter pores. Dynamic light scattering (DLS) analysis of the
liposomes composed of Zn2BDPA-PEG2000-DSPE–cholesterol–
POPC (2.5 : 30 : 67.5) indicated a monomodal size distribution
with diameter = 154 54 nm, PDI = 0.109, and ζ = 4.82 mV.
The same analysis of liposomes composed of Zn2BDPA-
PEG500-DSPE–cholesterol–POPC (2.5 : 30 : 67.5) indicated
a
bimodal size distribution with two diameters = 96 15 nm,
and 790 140 nm, PDI = 1.000, and ζ = 4.00 mV. We infer that
the larger diameter is due to liposome self-aggregation. Most
likely, the shorter PEG chain in the Zn2BDPA-PEG500-DSPE is
Scheme 2 Preparation of BDPA-PEG2000-DSPE (7). Conditions: (i)
PfpOH, EDC, DMAP, CHCl3, °C to rt, 16 followed by (ii)
H2N-PEG2000-DSPE, DIPEA, CHCl3, rt, 24 h.
0
h
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Scheme 3 Preparation of BDPA-PEG500-DSPE (10). Conditions: (i) PfpOH, EDC, DMAP, CHCl3, 0 °C to rt, 16 h followed by (ii) DSPE, DIPEA, CHCl3,
reflux, 24 h; (iii) piperidine–DMF 2 : 8, rt, 2 h; (iv) PfpOH, EDC, DMAP, CHCl3, 0 °C to rt, 16 h followed by (v) 9, DIPEA, CHCl3, rt, 24 h.
unable to fully block association of the terminal Zn2BDPA (10 : 30 : 60) resulted in rapid and extensive precipitation.
affinity units with the negatively charged phosphate diester Mixing with anionic liposomes composed of DPPG–choles-
groups on the opposing bilayer surface. Self-aggregation was terol–POPC (10 : 30 : 60) produced the same outcome (see
not observed with the liposomes containing Zn2BDPA-PEG2000
-
ESI†). In contrast, mixing with uncharged liposomes com-
DSPE, (four times longer PEG chain), therefore, all subsequent posed of cholesterol–POPC (30 : 70) produced no precipitation.
studies of Zn2BDPA coated liposomes used membrane com- Control experiments showed that the anionic liposome
positions containing this conjugated amphiphile.
systems do not form aggregates when exposed to Zn(NO3)2
The selective affinity of Zn2BDPA coated liposomes for alone, thus confirming that the Zn2BDPA affinity units are
anionic membranes was vividly demonstrated by conducting essential for the anionic membrane recognition process. These
experiments that mixed liposomes composed of Zn2BDPA- favorable liposome targeting results encouraged us to conduct
PEG2000-DSPE–cholesterol–POPC (2.5 : 30 : 67.5) with target cell recognition studies using both human and bacterial cells.
liposomes of various compositions. As shown in Fig. 2, mixing The liposomes were doped with a small amount of the near-
with anionic liposomes composed of POPS–cholesterol–POPC infrared fluorescent lipophilic dye, DiR, to enable effective
visualization using fluorescence microscopy.
Selective bacteria targeting using Zn2BDPA coated liposomes
The bacterial targeting of Zn2BDPA coated liposomes was eval-
uated by conducting imaging experiments using cultures of
E. coli, P. aeruginosa, S. aureus, and K. pneumoniae. In each
case, separate samples of bacteria (∼108 cells) were treated for
15 min with Zn2BDPA coated liposomes (Zn2BDPA-PEG2000
-
DSPE–DiR–cholesterol–POPC, 2 : 2 : 30 : 66) or untargeted lipo-
somes (DiR–cholesterol–POPC, 2 : 30 : 68). The treated cells
were pelleted by microcentrifugation and the tubes were
imaged using a CCD camera. As shown in Fig. 3, the fluore-
scent Zn2BDPA coated liposomes were located primarily in the
bacterial pellet, whereas, the fluorescent untargeted liposomes
were primarily in the supernatant above the pellet. After pellet
imaging, the bacterial cells were rinsed twice, dispersed
Fig. 2 Cross-linking of liposomes containing Zn2BDPA-PEG2000-DSPE into solution by vortexing, and subjected to fluorescence
and anionic target liposomes containing POPS. (A) Liposomes composed
of Zn2BDPA-PEG2000-DSPE–cholesterol–POPC (2.5 : 30 : 67.5 mol%,
0.83 µmol total lipid) in zinc-containing HEPES buffer. (B) Liposomes
composed of POPS–cholesterol–POPC (10 : 30 : 60 mol%, 0.83 µmol
microscopy. Shown in Fig. 4 are typical micrographs of cross-
linked bacteria/liposome aggregates. Analogous micrographs
of the pelleted bacteria that had been treated with fluorescent
untargeted liposomes showed no evidence for bacterial cross-
total lipid) in zinc-containing HEPES buffer. (C) Admixture of liposome
samples A and B.
linking (Fig. ESI-6†).
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Fig. 3 Four strains of pelleted bacteria after treatment with fluorescent
untargeted liposomes (left column) or fluorescent Zn2BDPA coated lipo-
somes (right column) and imaged using a CCD camera. (A) Brightfield;
(B) near-infrared fluorescence; (C) merge. 1 = E. coli, 2 = P. aeruginosa,
3 = S. aureus and 4 = K. pneumoniae. Scale bar indicates near-infrared
emission intensity for both fluorescence images in row B.
Fig. 5 Fluorescence micrographs of
a
mixture of DAPI stained
MDA-MB-211 human breast cancer cells, GFP-expressing P. aeruginosa,
and either near-infrared fluorescent untargeted liposomes (left column)
or near-infrared fluorescent Zn2BDPA coated liposomes (right column).
(row A) Brightfield images; (row B) Human cell nuclei stained with blue-
emitting DAPI; (row C) green-emitting P. aeruginosa; (row D) near-infra-
red fluorescence from Zn2BDPA coated liposomes; (row E) Overlay of
A, B, C and D. Scale bar = 30 µm.
Additional cell microscopy experiments demonstrated the
selectivity of the Zn2BDPA coated liposomes for bacterial cells
over healthy mammalian cells. Mixtures of near-infrared fluo-
rescent liposomes (coated with Zn2BDPA or untargeted),
MDA-MB-211 human breast cancer cells and GFP-expressing
P. aeruginosa bacteria were incubated for 15 min. As shown in
Fig. 5, only the mixtures with Zn2BDPA coated liposomes
resulted in bacterial agglutination. Fig. 5C and 5D show strong
co-localization of the near-infrared Zn2BDPA coated liposomes
and the green bacterial fluorescence, and no association of the
Zn2BDPA coated liposomes with the mammalian cell surfaces.
The untargeted liposomes did not interact with the bacteria
which remained widely dispersed across the entire micro-
graph. These results are consistent with our previous
Fig. 4 Representative fluorescence microscopy images of bacteria after
treatment with fluorescent Zn2BDPA coated liposomes. (left column)
Gram-positive S. Aureus bacteria, (right column) Gram-negative E. coli.
(Top row) Brightfield image of cross-linked bacteria/liposome aggre-
gate; (Middle row) Near-infrared fluorescence emission from Zn2BDPA
liposomes bound to the bacteria; (Bottom row) Overlay of brightfield
and fluorescence images. No fluorescence staining of bacterial cells was
observed with fluorescent untargeted liposomes. Scale bar = 30 µm.
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observations using multivalent molecular probes with four or PSVue480. Untargeted liposomes had no affinity for the dead
more covalently attached Zn2BDPA targeting units, and the and dying cell population.
independent work of others who have reported that magnetic
Zn2BDPA nanoparticles can be used to separate bacterial cells
from mammalian blood cells.48
Conclusion
The two amphiphilic conjugates, Zn2BDPA-PEG2000-DSPE and
Mammalian cell death targeting using Zn2BDPA coated liposomes
Zn2BDPA-PEG500-DSPE (Fig. 1C), were prepared using reliable
synthetic methods and incorporated into liposomes at low
molar fractions. Liposomes incorporating the shorter conju-
gate exhibited self-aggregation, whereas Zn2BDPA coated lipo-
somes incorporating the longer conjugate remained highly
dispersed in aqueous solution. The Zn2BDPA coated liposomes
formed cross-linked precipitates with target liposomes con-
taining anionic phospholipids, and did not interact with the
liposomes composed of uncharged phospholipids that were
mimics of the surfaces of healthy mammalian cells. The
Zn2BDPA coated liposomes selectively agglutinated bacterial
cells in the presence of healthy human cells. The selective
agglutination effect is most likely universal for both Gram-
positive and Gram-negative bacteria since it targets the anionic
amphiphiles in the bacterial envelope,49 and is complementary
to the highly specific cell recognition exhibited by antibodies
Standard MTT cell vitality assays showed that the Zn2BDPA
coated liposomes were not toxic to MDA-MB-231 human
breast cancer cells (Fig. ESI-7†). Furthermore, fluorescence
microscopy studies showed that fluorescently labeled Zn2BDPA
coated liposomes did not stain healthy MDA-MB-231 cells, but
they did stain dead and dying cells that had been treated with
a cytotoxic agent. Specifically, MDA-MB-211 cells were incu-
bated with etoposide (10 µM) followed by fluorescent
liposomes and PSVue480, a commercially available Zn2BDPA-
fluorescein molecular conjugate that has been validated as a
green-emitting fluorescent probe that targets exposed PS on
the exterior of dead and dying cells. As shown in Fig. 6, the
near-infrared Zn2BDPA coated liposomes co-localized with
which typically target
a specific antigenic protein. The
Zn2BDPA affinity unit is not bactericidal but multivalent
Zn2BDPA coated liposomes may have value as immobilization
agents that sequester an infection within an organism. Alter-
natively Zn2BDPA coated liposomes may have potential as anti-
biotic delivery vehicles.50,51 The selectivity for anionic cell
surfaces was further demonstrated by selective staining of
dead or dying mammalian cells in the presence of healthy
cells, an experimental outcome that is consistent with the tar-
geted liposome results of others.52–54 While Zn2BDPA coated
liposomes could simply be employed as dead cell stains for
in vitro assays, we envision a more ambitious in vivo appli-
cation as a cell death “theranostic” platform that combines
therapeutic and diagnostic capabilities within a single nano-
particle. Moreover, a liposomal system with both cell death
targeting and cytotoxic delivery capability has the potential of
producing amplified drug delivery to the dead and dying cells
within cancerous tissue.32,33
Experimental
Materials
FmocNH-PEG500-propionic acid (8) was obtained from Poly-
pure (Oslo, Norway). H2N-PEG2000-DSPE, 1-palmitoyl-2-oleoyl-
sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-
Fig. 6 Micrographs of human MDA-MB-211 cells treated with cytotoxic sn-glycero-3-phosphatidylserine (POPS), 1,2-dipalmitoyl-sn-
etoposide (10 μM) for 16 h and stained with green-emitting PSVue480
(10 μM), and either near-infrared fluorescent untargeted (left column) or
Zn2BDPA coated liposomes (right column). (row A) Brightfield; (row B)
Green-emission from cells stained with PSVue480; (row C) Near-infra-
glycero-3-phosphatidylglycerol (DPPG), were obtained from
Avanti Polar Lipids (Alabaster, Alabama, USA). The following
fluorescent molecular probes were purchased and used as sup-
plied PSVue480 (Molecular Targeting Technologies Inc.), DAPI
and DiR (Life Technologies).
red emission from cells stained with liposomes; (row D) Overlay of A, B,
and C. Scale bar = 30 µm.
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Synthesis
4.15 (q, J = 7.18 Hz, 2H), 4.64 (s, 4H) 6.82 (s, 2H) 6.91 (s, 1H).
13C NMR (75 MHz, CDCl3) δ 14.42, 24.79, 31.00, 60.80, 64.89,
Proton (1H-NMR) and carbon (13C-NMR) nuclear resonance 66.94, 112.11, 117.74, 143.03, 159.25, 173.72. HRMS (ESI+)
spectra were recorded on Varian UnityPlus 300 (300 MHz for calculated for C14H20NaO5 ([M
1H, 75 MHz for 13C), Varian INOVA 500 (500 MHz for 1H, 291.1203.
+
H]+) 291.1206; found
125 MHz for 13C), and Bruker Ascend 500 III HD (500 MHz for
Ethyl 4-(3,5-bis(bromomethyl)phenoxy)butanoate (4). To a
1H, 125 MHz for 13C) NMR spectrometers. High resolution solution of 170 mg (0.63 mmol) ethyl 4-(3,5-bis(hydroxy-
mass spectra were recorded on a Bruker micrOTOF II mass methyl)phenoxy)butanoate 415 mg (1.58 mmol) PPh3, and
spectrometer using electrospray ionization (ESI). Analytical 204 mg (1.58 mmol) DIPEA in 2 mL anhydrous THF was added
TLC was performed on EMD aluminum-backed 250 µm silica drop-wise 524 mg (1.58 mmol) CBr4 in 1 mL anhydrous THF.
gel 60 F254 and on EMD aluminum-backed 250 µm neutral The reaction was allowed to proceed at r.t. under argon for
aluminum oxide 60 F254 plates. Analytical TLC visualization 16 h. The reaction was quenched by addition of 10 mL satu-
was done under UV light (254 nm) or using conventional stain- rated NaBr. The organic solvent was removed under reduced
ing methods (I2, CAM, ninhydrin, sulfuric acid charring, and pressure. The residue was washed 3 times with 20 mL EtOAc.
so on). Preparatory scale TLC (P-TLC) was done using SiliCycle The combined organic phases were washed once with 30 mL
glass-backed 1000 and 2000 µm silica gel 60 F254 plates. Flash saturated NaBr and dried using MgSO4. The solvent was
column chromatography (FCC) was done by using either removed and the orange-brown residue subjected to flash
SiliCycle SiliaFlash P60 silica gel (40–63 µm, mesh 230–400) or column chromatography (SiO2, 1 : 1 EtOAc–Hex) to give 241 mg
Aldrich neutral aluminum oxide (Brockman I, 150 mesh, 58 Å). (97% yield) of product as a pale yellow oil. Rf = 0.48 (SiO2, 1 : 1
1
Anhydrous solvents for reactions were procured from commer- EtOAc–Hex). H NMR (300 MHz, CDCl3) δ 1.27 (t, J = 7.18 Hz,
cial sources except for THF (purified using an Innovative 3H), 2.12 (quin, J = 6.70 Hz, 2H), 2.53 (t, J = 7.29 Hz, 2H), 4.03
Technologies SPS-100-2 solvent purification system) and CHCl3 (t, J = 6.10 Hz, 2H), 4.16 (q, J = 7.18 Hz, 2H), 4.43 (s, 4H), 6.86
(distilled from P2O5).
(d, J = 1.20 Hz, 2H), 7.00 (s, 1H). 13C NMR (75 MHz, CDCl3)
(5-Hydroxy-1,3-phenylene)dimethanol (2). To a dispersion δ 14.50, 24.77, 30.92, 33.13, 60.75, 67.13, 115.39, 122.10,
of 1 g (26.25 mmol) LiAlH4 in 40 mL anhydrous THF at 0 °C 139.84, 159.46, 173.37. HRMS (ESI+) calculated for
was added drop-wise a solution of 3 g (14.27 mmol) dimethyl C14H18Br2O3 ([M + H]+) 392.9695; found 392.9718.
5-hydroxyisophthalate. The reaction was allowed to heat to
Ethyl
4-(3,5-bis((bis(pyridin-2-ylmethyl)amino)methyl)-
room temperature and stirred for 16 h. The reaction was phenoxy)butanoate (5). To a solution of 680 mg (1.73 mmol)
cooled to 0 °C and 20 mL 10% (v/v) H2SO4 was added drop- ethyl 4-(3,5-bis(bromomethyl)phenoxy)butanoate and 757 mg
wise. The solvent was removed under reduced pressure and (3.80 mmol) picolylamine in anhydrous DMF was added 1.2 g
residue was partitioned between 50 mL water and 50 mL (8.65 mmol) K2CO3. The reaction was stirred under Ar at r.t.
EtOAc. The aqueous phase was saturated with NaCl. The for 16 h. The solvent was removed under vacuum and the
aqueous phase was washed an additional 5 times using 50 mL residue partitioned between 20 mL each of water and CHCl3.
EtOAc each. The combined organic layers were washed once The aqueous phase was extracted 3 times using 20 mL CHCl3.
with 100 mL saturated NaCl and dried with MgSO4. The The combined organic phase was washed with 50 mL saturated
solvent was removed in vacuo to give 2.06 g (94% yield) of the NaCl, dried with MgSO4, and the solvent was removed. The
desired material as a tan solid. Rf = 0.28 (SiO2, EtOAc). 1H residue was subjected to flash column chromatography (Al2O3,
NMR: (500 MHz, CD3OD) δ 4.52 (s, 4H), 6.70 (s, 2H), 6.80 (s, CHCl3–MeOH 98 : 2) and 1.05 g of product (96% yield) was
1H). 13C NMR: (125 MHz, CD3OD) δ 63.95, 112.49, 116.44, obtained as a dark orange/brown oil. Rf = 0.77 (Al2O3, CHCl3–
1
143.13, 157.48. HRMS (ESI+) calculated for C8H10O3 ((M + H)+) MeOH 98 : 2). H NMR (300 MHz, CDCl3) δ 1.26 (t, J = 7.18 Hz,
155.0703, found 155.0673
Ethyl 4-(3,5-bis(hydroxymethyl)phenoxy)butanoate (3). To a 4H), 3.84 (s, 8H), 4.01 (t, J = 6.10 Hz, 2H), 4.15 (q, J = 7.18 Hz,
solution of 1 g (6.49 mmol) (5-hydroxy-1,3-phenylene)dimethan- 2H), 6.88 (s, 2H), 7.08 (s, 1H), 7.15 (ddd, J1 = 6.76 Hz, J2
3H), 2.11 (quin., J = 6.70 Hz, 2H) 2.53 (t, J = 6 Hz, 2H), 3.68 (s,
=
ol, 3.80 g (19.46 mmol) ethyl 4-bromobutyrate, and 120 mg 4.96 Hz, J3 = 1.91 Hz, 4H), 7.57–7.68 (m, 8H), 8.52 (d, J =
(0.32 mmol) tetrabutylammonium iodide in 15 mL anhydrous 4.78 Hz, 4H). 13C NMR (75 MHz, CDCl3) δ 14.47, 24.92, 31.10,
DMF was added 4.48 g (32.45 mmol) K2CO3. The reaction was 58.75, 60.22, 60.65, 66.89, 113.75, 121.78, 122.23, 123.00,
stirred at 60 °C under Ar for 16 h. The solvent was removed 136.69, 140.70, 149.17, 159.24, 159.78, 179.49. HRMS (ESI+)
under reduced pressure and the residue partitioned between calculated for C32H42N6O3 ([M
50 mL water and 50 ml EtOAc. The aqueous phase was washed 631.3423.
+
H]+) 631.3391, found
an additional 2 times with 50 ml EtOAc each. The combined
4-(3,5-Bis((bis(pyridin-2-ylmethyl)ammonio)methyl)phenoxy)-
organic phases were washed with 100 mL saturated NaCl and butanoate chloride (6). To a solution of 50 mg (77.5 μmol)
dried with MgSO4. Removal of the solvent and flash column ethyl 4-(3,5-bis((bis(pyridin-2-ylmethyl)amino)methyl)phenoxy)-
chromatography (SiO2, EtOAc) afforded 543 mg (∼31% yield) butanoate in 2 mL THF–Water–MeOH (2 : 1 : 1) was added
of the desired compound as a pale yellow oil. Rf = 0.40 (SiO2, 60 μl (0.34 mmol) 20% (w/w) NaOH (aq.). The reaction was
1
EtOAc). H NMR (300 MHz, CDCl3) δ 1.26 (t, J = 7.18 Hz, 3H) refluxed for 2 h, at which time TLC (Al2O3, CHCl3–MeOH
2.12 (m, 4H), 2.50 (t, J = 7.30 Hz, 2H), 4.01 (t, J = 6.10 Hz, 2H), 98 : 2) indicated complete conversion of the starting material.
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The pH of the reaction was adjusted to ∼7 by addition of product was eluted using 8 : 2 : 0.2 CHCl3–MeOH–H2O.
∼300 μl 1 M HCl. The solvents were removed under reduced Enriched fractions were pooled, the solvent was removed
pressure and the remaining solids were washed 3 times with under reduced pressure, the residue was dissolved minimal
10 mL CHCl3. The combined organic layers were dried with CHCl3, and was applied to a silica preparatory TLC plate which
MgSO4. Filtration and removal of solvent afforded ∼50 mg was developed using 8 : 2 : 0.2 CHCl3–MeOH–H2O to give
(∼99% yield) of the desired compound (BDPA-acid) as a pale 178 mg pure product. To remove ambiguity regarding the
yellow oil. Rf = 0.46 (Al2O3, CHCl3–MeOH–H2O 65 : 30 : 5); 0.27 counterion 150 mg of product was dissolved in 50 mL CHCl3
(SiO2, CHCl3–MeOH–NH4OH 8 : 2 : 0.2). 1H NMR (500 MHz, and washed 2 times with 50 mL saturated NaCl which was
CD3OD) δ 2.05 (quin., 2H, J = 6.72 Hz), 2.47 (t, 2H, J = 7.21 Hz), acidified to pH 4. The organic phase was dried using Na2SO4
3.63 (s, 4H), 3.77 (s, 8H), 4.00 (t, J = 6.36 Hz, 2H), 6.83 (s, 2H), and removal of solvent in vacuo yielded 146 mg (∼65% yield) of
6.99 (s, 1H), 7.21–7.28 (m, 4H), 7.64 (d, J = 7.83 Hz, 4H), 7.76 FmocNH-PEG500-DSPE. Rf = 0.46 (SiO2, CHCl3–MeOH–H2O
1
(td, J = 7.70 Hz, J = 1.71 Hz, 4H), 8.31–8.46 (m, 4H). 13C NMR 8 : 2 : 0.2). H NMR (300 MHz, CDCl3) δ 0.88 (t, 3H), 1.19–1.39
(125 MHz, CD3OD) δ 24.80, 30.56, 58.69, 59.70, 66.94, 114.01, (m, ∼60H), 1.58 (m, 5H), 2.28 (td, J = 7.53, 2.63 Hz, 4H), 2.5 (t,
121.77, 122.61, 123.54, 137.49, 140.16, 148.22, 159.22, 159.37, J = 5.50 Hz, 2H), 3.26–3.50 (m, 5H), 3.50–3.82 (m, ∼56H), 3.88
176.07. HRMS (ESI+) calculated for C36H39N6O3 ([M + H]+) (t, J = 6.22 Hz, 1H), 4.00 (t, J = 5.86 Hz, 4H), 4.08–4.30 (m, 3H),
603.3078; found 603.3067.
4.31–4.50 (m, 4H), 5.17–5.27 (m, 1H), 5.53 (m, 1H), 7.28–7.35
BDPA-PEG2000-DSPE (7). To
a
solution of 17.3 mg (m, 2H), 7.41 (t, J = 7.18 Hz, 2H), 7.61 (d, J = 7.41 Hz, 2H), 7.77
(0.027 mmol) BDPA-acid (6) and 5 mg pentafluorophenol (d, J = 7.41 Hz). 13C NMR (75 MHz, CDCl3) δ 14.36, 15.34,
(0.027 mmol) in 1.7 mL anhydrous CHCl3 under argon at 0 °C 22.92, 25.08, 29.36, 29.59, 29.94, 32.14, 34.26, 34.41, 35.43,
was added drop wise 5.2 mg (0.027 mmol) EDC and 0.66 mg 36.56, 41.13, 42.76, 47.46, 62.41, 66.75, 67.43, 70.05, 70.26,
(0.0054 mmol) DMAP each in 0.1 mL anhydrous CHCl3. The 70.37, 70.75, 120.16, 125.28, 127.25, 127.86, 141.50, 144.19,
reaction was kept at 0 °C for 30 min and then allowed to warm 172.59, 173.20, 173.57, 173.62. HRMS (ESI+) calculated for
to room temperature and stirred for 16 h. 30 mg (0.011 mmol) C83H146N2O23P− ((M + 2H)+) 1569.0054, found 1568.9970.
H2N-PEG2000-DSPE and 3.5 mg (4.7 µl, 0.011 mmol) DIPEA in 146 mg (91.8 µmol) of FmocNH-PEG500-DSPE was dissolved in
0.1 mL anhydrous CHCl3 was added to the reaction and stirred 5 mL 20% piperidine in DMF. The reaction was allowed to
at room temperature under argon for 24 h. The crude reaction proceed for 2 h at room temperature, after which the solvent
mixture was subjected to preparatory scale TLC on silica using was removed in vacuo and the residue was subjected to flash
8 : 2 : 0.2 CHCl3–MeOH–NH4OH as the eluent followed by a column chromatography (SiO2, CHCl3–MeOH–water 8 : 2 : 0.2)
second P-TLC run on SiO2 using 8 : 1 : 0.1 CHCl3–MeOH– 4 times to yield enriched product. This material was purified
NH4OH as the eluent to give 24 mg (∼65% yield) of product as after two runs on preparatory TLC (SiO2, CHCl3–MeOH–H2O
a yellow/orange solid film. Rf = 0.25 (SiO2, 8 : 1 : 0.1 CHCl3– 8 : 2 : 0.2). 90 mg (∼73% yield) of product was obtained as a
1
MeOH–NH4OH). H NMR (300 MHz, CDCl3, CD3OD) δ 0.84 (t, white solid. Rf = 0.26 (SiO2, CHCl3–MeOH–H2O 8 : 2 : 0.2). 1H
J = 6 Hz, 6H), 1.21 (m, ∼65H), 1.54 (m, 4H), 2.07 (m, 2H), 2.24 NMR (300 MHz, CDCl3) δ 0.88 (t, 6H), 1.26 (m, ∼58H), 1.59 (m,
(td, J1 = 7.53 Hz, J2 = 2.39 Hz, 4H), 2.36 (m, 2H), 3.38 (m, 4H), 4H), 2.29 (td, J = 7.59, 3.95 Hz, 5H), 2.50 (t, J = 6.10 Hz, 2H),
3.44–3.76 (m, ∼216H), 3.85 (m, 10H), 3.95 (m, 6H), 4.07–4.22 3.19 (m, 2H), 3.38–3.52 (m, 3H), 3.53–3.86 (m, ∼51H),
(m, 4H), 4.34 (dd, J1 = 12.08 Hz, J2 = 3.23 Hz, 1H), 5.17 (m, 3.94–4.09 (m, 2H), 4.17 (dd, J = 12.08, 6.58 Hz, 1H), 4.40 (dd,
1H), 6.85 (s, 2H), 7.03 (s, 1H), 7.13–7.20 (m, 4H), 7.54–7.71 (m, J = 11.96, 3.35, 1H), 5.22 (m, 1H). 13C NMR (75 MHz, CDCl3)
8H), 8.45 (d, J = 4.54 Hz, 4H). 13C NMR (125 MHz, CDCl3, δ 13.69, 22.51, 24.74, 24.77, 29.00, 29.18, 29.22, 29.51, 29.55,
CD3OD) δ 14.25, 22.84, 25.04, 25.07, 25.50, 29.32, 29.51, 29.81, 31.79, 33.91, 34.04, 36.29, 39.42, 62.18, 64.18, 64.88, 66.64,
29.86, 32.08, 32.87, 34.27, 34.44, 39.32, 42.41, 58.72, 59.69, 66.99, 69.52, 69.66, 69.73, 69.83, 69.90, 69.97, 70.09,
62.81, 63.57, 63.68, 64.42, 67.27, 69.89, 70.02, 70.23, 70.38, 70.17, 70.23, 173.35, 173.75. HRMS (ESI+) calculated for
70.63, 70.69, 114.18, 121.89, 122.55, 123.34, 137.23, 139.96, C68H134N2O21P− ((M + 2H)+) 1347.9368, found 1347.9354; MS
148.77, 158.83, 159.39, 173.32, 173.72. HRMS (ESI+) calculated (ESI+) calculated for C68H134N2O21P− ((M
+
H
+
Na)+)
for
C
168H298N8O56P− ([(M
+
3H)/2]+) 1679.0339, found 1369.9187, found 1369.9177.
1679.5384.
BDPA-PEG500-DSPE (10). To
a
solution of 88.9 mg
NH2-PEG500-DSPE (9). To a solution of 146 mg (0.174 mmol) (0.139 mmol) BDPA-acid (6) and 25.6 mg pentafluorophenol
8 in 5 mL anhydrous CHCl3 at 0 °C under argon was sequen- (0.139 mmol) in 3 mL anhydrous CHCl3 under argon at 0 °C
tially added 48 mg (0.261 mmol) pentafluorophenol, 50 mg was added drop wise 26.6 mg (0.139 mmol) EDC and 3.33 mg
(0.261 mmol) EDC, and 4.3 mg (34.8 µmol) DMAP. The reac- (0.0273 mmol) DMAP each in 0.5 mL anhydrous CHCl3. The
tion was kept at 0 °C for 30 min, then allowed to warm to reaction was kept at 0 °C for 30 min and then allowed to
room temperature and stirred for 16 h. To this reaction warm to room temperature and stirred for 16 h. 80 mg
mixture was added 169 mg (0.226 mmol) DSPE followed by (0.0594 mmol) H2N-PEG500-DSPE and 19.2 mg (25.9 µl,
33.7 mg (45.5 µl, 0.261 mmol) DIPEA in 1 mL anhydrous 0.149 mmol) DIPEA in 0.5 mL anhydrous CHCl3 was added to
CHCl3. The reaction was refluxed for 24 h after which the the reaction and stirred at room temperature under argon for
solvent was removed in vacuo. The residue was dissolved in 24 h. The crude reaction mixture was subjected to preparatory
minimal CHCl3 and applied to a silica flash column and the scale TLC on silica using 8 : 2 : 0.2 CHCl3–MeOH–NH4OH as
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the eluent followed by a second P-TLC run on SiO2 using insertions, gift from Professor J. Shrout) were grown to mid-log
8 : 1 : 0.1 CHCl3–MeOH–NH4OH as the eluent to give 100 mg phase (OD = 0.5) in Luria Bertani (LB) broth (5 g L−1 yeast
(∼86% yield) of product as an orange/brown solid film. Rf = extract, 10 g L−1 Tryptone, 10 g L−1 NaCl) at 37 °C and a
0.50 (SiO2, 8 : 2 : 0.2 CHCl3–MeOH–NH4OH). 1H NMR shaker speed of 200 rpm. The samples were centrifuged (5000
(300 MHz, CDCl3) δ 0.87 (t, J = 6 Hz, 6H), 1.25 (m, 60H), 1.57 rpm, 5 min) and the pellet resuspended in 1 mL of HEPES
(m, 4H), 2.11 (m, 2H), 2.27 (m, 4H), 2.41 (t, J = 6 Hz, 2H), 2.48 buffer (10 mM HEPES, 145 mM NaCl, pH = 7.4). Each sample
(t, J = 6 Hz, 2H), 3.45 (m, 4H), 3.50–3.79 (m, 59H), 3.86 (s, 8H), of bacteria (∼108 cells) was treated for 15 min with near-infra-
3.99 (m, 6H), 4.17 (dd, J1 = 11.96 Hz, J2 = 6.70 Hz, 1H), 4.39 red fluorescent Zn2BDPA coated liposomes (Zn2BDPA-PEG2000
-
(dd, J1 = 12.20 Hz, J2 = 3.11 Hz, 1H), 5.23 (m, 1H), 6.88 (s, 2H), DSPE–DiR–cholesterol–POPC, 2 : 2 : 30 : 66) or untargeted lipo-
7.06 (s, 1H), 7.17 (m, 4H), 7.58–7.71 (m, 8H), 8.53 (d, J = 5.02 somes (DiR–cholesterol–POPC, 2 : 30 : 68). The treated cells
Hz, 4H). 13C NMR (75 MHz, CDCl3) δ 14.10, 22.66, 24.85, were pelleted by microcentrifugation at 5000 rpm for 5 min
24.89, 25.28, 29.11, 29.30, 29.33, 29.63, 29.68, 31.89, 32.76, and the tubes were imaged using a CCD camera within a
34.07, 34.25, 36.74, 39.15, 58.48, 59.50, 62.65, 66.98, 67.45, Xenogen imaging station. A region of interest analysis of the
69.84, 70.07, 70.10, 70.43, 113.72, 122.11, 122.95, 136.11, fluorescence images indicated that the pellets contained 98%
148.72, 159.07, 171.36, 172.48, 172.99, 173.37. HRMS (ESI−) and 6% of the Zn2BDPA coated liposomes and untargeted lipo-
calculated for
1931.2042.
C
104H170N8O23P− (M−) 1931.2155, found somes, respectively. The bacterial cells were washed twice with
buffer to reduce background fluorescence, dispersed into solu-
tion then onto slides, and micrographs were acquired using a
Nikon Eclipse TE2000-U epifluorescence microscope with a
Liposome studies
Liposome preparation for cuvette experiments. All polar 60× objective and a Photometrics Cascade 512B CCD. Near
lipids were purchased from Avanti Polar lipids (Alabaster, AL, infrared fluorescence images were captured using a Cy7 filter
USA) and were used without further purification. Lipid films set (Exciter HQ710/75x, Dichroic Q750LP, Emitter HQ810/
consisting of appropriate molar fractions of polar lipid (3.32 90m).
µmol total lipid) were prepared by dispensing measured ali-
Fluorescence microscopy was used to demonstrate the
quots from stock chloroform solutions, evaporating the selectivity of the Zn2BDPA coated liposomes for bacterial cells
solvent under a gentle stream of argon, and placing the films over healthy mammalian cells. The nuclei of confluent
under vacuum overnight to ensure complete removal of MDA-MB-231 human breast cancer cells were stained by incu-
solvent. The films were hydrated by adding 1 mL of HEPES bating with DAPI (1 μg mL−1 DAPI in PBS) for 30 minutes.
buffer (pH 7.4, 10 mM HEPES, 137 mM NaCl, 3.2 mM KCl, Separate samples of the DAPI stained MDA-MB-231 cells were
2 mM NaN3) or in some cases a 1 mL solution of HEPES buffer washed with HEPES buffer then treated with near-infrared
containing Zn(NO3)2. The hydrated lipid films were subjected fluorescent Zn2BDPA coated liposomes or untargeted lipo-
to six freeze–thaw cycles (40 °C ↔ liquid N2) and then passed somes in HEPES buffer. An aliquot of GFP-expressing P. aerugi-
21 times through a 19 mm polycarbonate Nucleopore filter nosa (20 µL from a broth grown to an OD600 = 0.2) was added
with 200 nm diameter pores using an Avestin LiposoFast mini to the samples, which were incubated for 15 minutes then
extruder to produce unilamellar liposomes. Aliquots (125 µL) imaged using fluorescence microscopy. Fluorescence images
of Zn2BDPA coated liposomes were diluted by adding HEPES were captured using UV (ex: 340/80, em: 435/85), GFP (ex: 450/
buffer (875 µL) and subjected to dynamic light scattering 90, em: 500/50), or Cy7 filter sets.
(DLS) analysis using a Zetasizer Nano ZS particle sizer (25 °C,
173.1° backscatter angle with appropriate material, dispersant,
Mammalian cell toxicity
and RI parameters selected). Liposome cross-linking exper- Toxicity of the liposomal formulations was measured using the
iments mixed 500 µL of HEPES buffer with 250 µL of lipo- 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide
somes composed of Zn2BDPA-PEG2000-DSPE–cholesterol–POPC (MTT) cell vitality assay. MDA-MB-231 human breast cancer
(2.5 : 30 : 67.5) and 250 µL of target liposomes with various cells (ATCC) were seeded into 96-microwell plates, and grown
compositions. The results were photographed using a digital to a confluency of 85% in RPMI media with 10% fetal bovine
camera.
serum, and 1% streptavidin L-glutamate at 37 °C and 5% CO2.
Fluorescent liposome preparation for cell studies. Lipo- The Vybrant MTT Cell Proliferation Assay Kit (Invitrogen,
somes containing 2 mol% 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl- Eugene, USA) was performed according to the manufacturer’s
indotricarbocyanine iodide (DiR) were prepared using the protocol and validated using 10 µM etoposide as a positive
thin film hydration method described above, and a zinc- control for high toxicity. The cells were treated with untargeted
containing hydration buffer (10 mM HEPES, 145 mM NaCl, liposomes
(cholesterol–POPC,
30 : 70)
and
Zn2BDPA
100 µM ZnNO3, pH = 7.4). coated liposomes (Zn2BDPA-PEG2000-DSPE–cholesterol–POPC,
Bacterial agglutination studies. Samples of Staphylococcus 2 : 30 : 68) and incubated for 18 h at 37 °C. The medium was
aureus NRS11 (gift from Professor S. Mobashery), Escherichia removed and replaced with 100 μL of RPMI media containing
coli UTI89 (gift from Professor D. Piwnica-Worms), Klebsiella MTT (1.2 mM). An SDS detergent solution was added and incu-
pneumoniae (ATCC #33495), and Pseudomonas aeruginosa (con- bated at 37 °C and 5% CO2 for an additional 4 hours. The
taining mini-Tn7 chromosomal, constitutive, GFP-expressing absorbance of each well was read at 570 nm and the normalized
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Organic & Biomolecular Chemistry
data (measured in triplicate) are shown in Fig. ESI-7.† The
results indicate that the untargeted and Zn2BDPA coated lipo-
D. O. Kiesewetter, S. Lee and X. Chen, Angew. Chem., Int.
Ed., 2012, 51, 445–449.
somes induce negligible amounts of cell death at concen- 14 K. Honda, S. H. Fujishima, A. Ojida and I. Hamachi, Chem-
trations <10 µM and <25 µM respectively.
BioChem, 2007, 8, 1370–1372.
15 A. Ojida, K. Honda, D. Shinmi, S. Kiyonaka, Y. Mori and
I. Hamachi, J. Am. Chem. Soc., 2006, 128, 10452–10459.
16 K. Honda, E. Nakata, A. Ojida and I. Hamachi, Chem.
Commun., 2006, 4024–4026.
17 J. A. Drewry, S. Burger, A. Mazouchi, E. Duodu, P. Ayers,
C. C. Gradinaru and P. T. Gunning, MedChemComm, 2012,
3, 763–770.
18 J. R. Morrow and O. Iranzo, Curr. Opin. Chem. Biol., 2004, 8,
192–200.
19 V. Ganesh, K. Bodewits, S. J. Bartholdson, D. Natale,
D. J. Campopiano and J. C. Mareque-Rivas, Angew. Chem.,
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Mammalian cell death imaging using fluorescent liposomes
Confluent MDA-MB-211 cells were treated with etoposide
(10 µM) for 16 hours to induce apoptotic cell death. Separate
populations of apoptotic MDA cells were treated with near-
infrared fluorescent Zn2BDPA coated liposomes or untargeted
liposomes in HEPES buffer and allowed to incubate for
15 minutes. The cells were then washed three times followed
by treatment with green-emitting cell death probe PSVue480
(10 μM) for 15 minutes and three subsequent wash steps.
Fluorescence micrographs were captured using the microscope
described above and a GFP or Cy7 filter set.
20 E. Kinoshita, M. Takahashi, H. Takeda, M. Shiro and
T. Koike, Dalton Trans., 2004, 1189–1193, DOI: 10.1039/
b400269e.
21 L. Ciavatta, J. C. Mareque, D. Natale and F. Salvatore, Ann.
Chim., 2006, 96, 317–325.
22 A. V. Koulov, K. A. Stucker, C. Lakshmi, J. P. Robinson and
B. D. Smith, Cell Death Differ., 2003, 10, 1357–1359.
23 A. V. Koulov, R. G. Hanshaw, K. A. Stucker, C. Lakshmi and
B. D. Smith, Isr. J. Chem., 2005, 45, 373–379.
24 R. G. Hanshaw, C. Lakshmi, T. N. Lambert, J. R. Johnson
and B. D. Smith, ChemBioChem, 2005, 6, 2214–2220.
25 W. M. Leevy, S. T. Gammon, J. R. Johnson, A. J. Lampkins,
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Acknowledgements
This study was supported by the NIH (RO1GM059078 to B.D.S.
and T32GM075762 to D.R.R.), Walther Cancer Research Foun-
dation, the University of Notre Dame, the Notre Dame
Integrated Imaging Facility, and the Freimann Life Sciences
Center. S.T. gratefully acknowledges support from the Scienti-
fic and Technological Research Council of Turkey (Grant
number 114C041).
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