Non-Covalent Assembly Method that Simultaneously Endows a Liposome Surface with Targeting Ligands, Protective PEG Chains, and Deep-Red Fluorescence Reporter Groups

Article abstract of DOI:10.1002/chem.201702649

A new self-assembly method is used to rapidly functionalize the surface of liposomes without perturbing the membrane integrity or causing leakage of the aqueous contents. The key molecule is a cholesterol-squaraine-PEG conjugate with three important structural elements: a cholesterol membrane anchor, a fluorescent squaraine docking station that allows rapid and high-affinity macrocycle threading, and a long PEG-2000 chain to provide steric shielding of the decorated liposome. The two-step method involves spontaneous insertion of the conjugate into the outer leaflet of pre-formed liposomes followed by squaraine threading with a tetralactam macrocycle that has appended targeting ligands. A macrocycle with six carboxylates permitted immobilization of intact fluorescent liposomes on the surface of cationic polymer beads, whereas a macrocycle with six zinc(II)-dipicolylamine units enabled selective targeting of anionic membranes, including agglutination of bacteria in the presence of human cells.

Full text of DOI:10.1002/chem.201702649

A Journal of
Accepted Article
Title: Non-Covalent Assembly Method that Simultaneously Endows
a Liposome Surface with Targeting Ligands, Protective PEG
Chains, and Deep-Red Fluorescence Reporter Groups
Authors: Scott K Shaw, Wenqi Liu, Seamus Brennan, Maria
Betancourt-Mendiola, and Bradley D. Smith
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201702649
Link to VoR: http://dx.doi.org/10.1002/chem.201702649
Supported by

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Non-Covalent Assembly Method that Simultaneously Endows a
Liposome Surface with Targeting Ligands, Protective PEG
Chains, and Deep-Red Fluorescence Reporter Groups
Scott K. Shaw, Wenqi Liu, Seamus P. Brennan, María de Lourdes Betancourt-Mendiola, and Bradley
D. Smith*[a]
strategy.[10] The simplest way to fabricate targeted liposomes is to
disperse a pre-mixed film containing the various membrane
components. However this method results in unwanted
decoration of the liposome inner monolayer, which wastes
valuable targeting components and also may complicate the
subsequent loading of liposome payload.[11] Ideally, surface
functionalization should be done after liposome fabrication and
payload incorporation. Most studies that decorate the surface of
liposomes use covalent conjugation chemistry, and while useful
targeted systems have been developed, the bimolecular reactions
are relatively slow and it is hard to ascertain if and when they have
reached completion.[12–20] This is potentially problematic because
it leaves reactive groups on the liposome surface which could
abrogate selective targeting, thus requiring an additional chemical
capping step.[21] Rapid surface functionalization is highly desirable
with pre-formed liposomes that are filled with payload since it
minimizes the time period for unwanted background leakage of
the payload. In principle, the liposome exterior could be rapidly
decorated using non-covalent assembly methods that have
inherently low activation barriers, but progress has been limited,
in large part because there are very few association systems that
have the desired combination of supramolecular properties. Two
Abstract: A new self-assembly method is used to rapidly
functionalize the surface of liposomes without perturbing the
membrane integrity or causing leakage of the aqueous contents.
The key molecule is a cholesterol-squaraine-PEG conjugate with
three important structural elements: a cholesterol membrane
anchor, a fluorescent squaraine docking station that allows rapid
and high-affinity macrocycle threading, and a long PEG-2000
chain to provide steric shielding of the decorated liposome. The
two-step method involves spontaneous insertion of the conjugate
into the outer leaflet of pre-formed liposomes followed by
squaraine threading with a tetralactam macrocycle that has
appended targeting ligands. A macrocycle with six carboxylates
permitted immobilization of intact fluorescent liposomes on the
surface of cationic polymer beads, whereas a macrocycle with six
zinc(II)-dipicolylamine units enabled selective targeting of anionic
membranes, including agglutination of bacteria in the presence of
human cells.
Introduction
early
approaches,
using
biotin/(strept)avidin[22]
or
polyhistidine/Ni(II) chelate[23] as the affinity partners to attach
proteins to liposomes, were shown to have various performance
drawbacks. More recent designs, using liposomes with
embedded container molecules such amphiphilic cyclodextrin[24]
or calixarene[25] derivatives as docking sites for targeting ligands,
have produced interesting in vitro results but are likely to have
limited in vivo utility due to the modest association constants.
In recent years we have developed a new non-covalent,
self-assembly process that we call Synthavidin (Synthetic
Avidin).[26] In short, a deep-red fluorescent squaraine dye is
encapsulated by a water-soluble tetralactam macrocycle to give
an extremely stable, threaded complex that is well suited for
fluorescence imaging. To date, we have pre-assembled
fluorescent molecular probes that selectively target bone,
dead/dying mammalian tissue, and various microbial
species.[27,28] One of the major advantages of Synthavidin self-
assembly is the capability to form threaded complexes with long
PEG chains appended to the ends of the squaraine dye.[29,30] This
means the length of the PEG chains is a structural parameter that
can be systematically varied in order to optimize the
pharmacokinetic properties of the pre-assembled probes. In this
report we describe for the first time how Synthavidin self-
assembly can be exploited as a new and generalizable non-
covalent method to functionalize the surface of pre-formed
Liposomes have been investigated extensively for use as
nanocapsules for nanotechnology, drug delivery and molecular
imaging.[1,2] The first generation of therapeutic liposomes to
receive clinical approval were non-targeted carriers of
a
pharmaceutical payload,[3] and building on this success is an
ongoing effort to develop next generation surface-functionalized
nanoparticles for cell or tissue selective targeting.[4–6] The design
of targeted liposomes must satisfy several requirements for
successful operation in living subjects.[7] The liposome exterior
must contain multiple copies of an effective targeting agent, but it
also must be protected by long hydrophilic polymers such as
polyethylene glycol (PEG) that stabilize the colloidal particles and
block undesired uptake by the reticuloendothelial system
(RES).[8,9] In addition, there is great value gained by incorporating
a reporter group into the liposome structure to permit imaging
experiments that can assess the success of the targeting
[a]
S. K. Shaw, W. Liu, S. P. Brennan, Dr. M. L. Betancourt-Mendiola,
Dr. B. D. Smith
Department of Chemistry & Biochemistry
University of Notre Dame
236 Nieuwland Science Hall, University of Notre Dame, IN., 46545,
USA.
E-mail: smith.115@nd.edu
liposomes.
A summary of the two-step liposome surface
Supporting information for this article is given via a link at the end of
the document.
functionalization method is shown in Scheme 1. The central
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Scheme 1. Summary of two-step method for surface functionalization of POPC liposomes.
molecule is the newly designed cholesterol-squaraine-PEG
conjugate, CSP, which has three important structural elements: a
cholesterol membrane anchor, a fluorescent squaraine docking
station that allows rapid and high-affinity macrocycle threading,
and a long PEG-2000 chain to provide steric shielding of the
decorated liposome. The first step in the method is spontaneous
insertion of the CSP conjugate into the outer leaflet of pre-formed
liposomes without disturbing the bilayer. In step two the squaraine
component of the anchored CSP is threaded with a tetralactam
macrocycle that has appended targeting ligands. The study
employed two different tetralactam macrocycles, 6C with six
carboxylates which enhance liposome affinity for cationic
surfaces, and 6Z with six zinc(II)-dipicolylamine (ZnDPA) units
which enable selective targeting of anionic membranes and cell
surfaces.[31] A major attribute of this surface functionalization
method is its efficiency, as it simultaneously endows the
liposomes with targeting ligand, protective PEG chains, and deep-
red fluorescence reporter group. A potential concern at the start
of the study was whether the surrounding corona of PEG chains
would attenuate the targeting capabilities of the sheltered ligands.
The limited literature on this broadly important question
suggested that the outcome was context dependent.[21,32–34]
squaraine absorption band, quenched fluorescence and
observation of ~7 nm micelles using dynamic light scattering
(Figures 1A and S1). Mixing an aqueous solution of self-
aggregated CSP with an aliquot of pre-formed, unilamellar 1-
palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC)
liposomes (200 nm diameter) resulted in fluorescence turn-on as
the CSP inserted into the membrane outer leaflet to create
POPC@CSP liposomes. This membrane insertion step was
complete within 60 s (Figure 1B) and the squaraine fluorescence
remained with the liposomes after they were filtered through a
size exclusion column, indicating that the CSP was embedded in
the liposomes. This conclusion was confirmed by separates
experiments that inserted CSP into POPC liposomes containing
the lipophilic fluorescence resonance energy transfer (FRET)
partner DiI and observing FRET from the DiI donor to CSP
acceptor (Figure S2). The demonstration that CSP inserts
strongly into the liposomes is important since recent literature
indicates that not all cholesterol-based amphiphiles are well
anchored in bilayer membranes.[35,36,37]
Step two of the liposome surface functionalization method is
macrocycle threading of the squaraine dye that projects from the
exterior of POPC@CSP liposomes. The feasibility of this
threading step was initially tested using the multianionic
macrocycle, 6C, which exhibits “ideal” supramolecular behaviour
Results and Discussion
The CSP conjugate was synthesized in straightforward
fashion by conducting two sequential azide/alkyne cycloaddition
reactions that attached azido-cholesterol and azido-PEG-2000
components to each end of a central squaraine bis(alkyne) dye
(Scheme S2). The squaraine dye has two important structural
features: (a) its C2O4 core is flanked by two 2-aminothiophene
rings which donate electron density to the core and provide high
chemical stability in aqueous solution, and (b) an N-methyl group
at the end of the dye ensures rapid and high-affinity macrocycle
threading.[30] As a free compound, the CSP conjugate is soluble
in water but self-aggregated, as indicated by a broadened
Figure 1. (A) Fluorescence spectra (ex: 630 nm) of CSP (1 µM) in water or after
insertion into POPC liposomes. (B) Change in CSP fluorescence (ex: 630 nm,
em: 680 nm) over time after addition of POPC liposomes and waiting ~10 s. In
both panels, CSP was 0.3 mol% respective to POPC.
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proved that there was no translocation of liposome-embedded
⊃
CSP or the CSP 6C complex across the bilayer membrane. This
was achieved by comparing the absorption and fluorescence
spectra for a series of liposome dispersions with the following sets
of labelling conditions: (a) CSP or CSP
only in the membrane outer leaflet (Figure S5), (b) CSP or
CSP 6C complex embedded equally in both membrane leaflets,
and (c) CSP 6C complex only in the membrane outer leaflet and
CSP only in the membrane outer leaflet (Figure S6). The spectra
showed that the fraction of CSP or CSP 6C complex in each
⊃
6C complex embedded
⊃
⊃
⊃
membrane leaflet was unchanged over time. Analyses using
dynamic light scattering (DLS) indicated that the average
liposome size did not change during the two-step surface
functionalization process to make the POPC@CSP 6C
⊃
liposomes (Figure 2D). Additionally, liposome leakage
experiments revealed that the surface functionalization procedure
induced no significant release of liposome aqueous contents
(chloride anions) (Figure S7).
In order to prepare a cell targeting system, we chose to
decorate the liposome surface with ZnDPA units. Fluorescence
titration experiments mixed POPC@CSP liposomes with the
macrocycle 6Z (appended with six ZnDPA units) to produce
Figure 2. Fluorescence excitation/emission spectra of; (A) POPC@CSP
liposomes and (B) POPC@CSP⊃6C liposomes. (C) Fluorescence titration that
added 6C to POPC@CSP liposomes and monitored increase in fluorescence
intensity (F/Fo, ex: 690 nm, em: 700 nm) due to formation of
targeted multivalent POPC@CSP
the preparation of POPC@CSP
⊃
6Z liposomes. Consistent with
POPC@CSP⊃6C. (D) Dynamic light scattering showed that liposome surface
functionalization produced no change in liposome size. In all panels, the CSP
was 0.3 mol% respective to POPC.
⊃
6C liposome described above,
the threading of 6Z was indicated by a red-shift in squaraine
absorption/emission bands and the Ka was determined to be (2.4
± 0.4) × 108 M-1 (Figure S8C). Additional experiments showed that
the two-step surface functionalization process to produce
(i.e., the multianionic macrocycle and its threaded complexes
have no tendency to self-aggregate) while conferring the
liposome with the ability to target cationic surfaces. Shown in
POPC@CSP 6Z liposomes caused no change in liposome size
⊃
(Figure S8D) and no significant leakage of liposome aqueous
contents (sulforhodamine dye) (Figure S13). Furthermore, the
⊃
embedded CSP 6Z complex remained in the liposome outer
Figure
POPC@CSP liposomes were treated with tetralactam
macrocycle 6C to produce POPC@CSP 6C liposomes. The
2 A-B are the spectral changes observed when
⊃
leaflet and did not transfer over time to other liposomes in the
sample (Figure S9) or translocate to the inner leaflet of the
liposome membrane (Figure S10-S11). However, the threading to
threading process was indicated by diagnostic ~20-40 nm red-
shifts in the deep-red squaraine absorption/fluorescence maxima
(Table S2), and formation of the threaded complex was confirmed
by observing efficient fluorescence energy transfer from the
anthracene sidewalls of the surrounding macrocycle (ex: 390 nm)
to the encapsulated squaraine. The threading-induced red-shift in
squaraine emission maxima made it easy to conduct fluorescence
titration experiments that added aliquots of 6C to POPC@CSP
liposomes where the loading of CSP was 0.3 mol% respective to
POPC. Macrocycle threading was complete within seconds after
each aliquot addition, and as shown in Figure 2C the titration
isotherms fitted well to a 1:1 association model. A remarkably high
Ka of (2.8 ± 0.2) × 108 M-1 was determined which is quite close to
the Ka previously determined for threading of 6C by a water-
soluble analogue of CSP (Scheme S3 and Table S1) and shows
that the surface of the POPC@CSP liposomes does not inhibit
squaraine threading of 6C.[38] Passage of the surface
produce POPC@CSP 6C and POPC@CSP 6Z liposomes
⊃ ⊃
differed in two major ways. One was the relatively slow rate of 6Z
threading on the surface of the POPC@CSP liposomes. As
shown in Figure 3A the threading of 6C was complete within a few
seconds, whereas the threading of 6Z required ~20 minutes
(Figure 3A). Previous studies have shown that the threading of 6Z
by a dispersed, water soluble squaraine is fast[28] so the slow
formation of POPC@CSP 6Z must be a liposome surface effect.
⊃
The most likely explanation is surface promoted association of the
functionalized POPC@CSP 6C liposomes through a size
⊃
exclusion column did not lower the fluorescence signal, indicating
that the threaded complex remained embedded in the liposomes.
Furthermore, a series of FRET experiments using liposomes with
embedded DiI as a FRET partner confirmed that there was no
⊃
transfer of the threaded complex out of the POPC@CSP 6C
liposomes into a large excess of neighbouring liposomes (Figures
S2-S4). A series of comparative fluorescence experiments also
Figure 3. (A) Kinetics of association as measured by fluorescence (ex: 685
nm em: 710 nm). Threading by 6C occurred in seconds while complete
threading by 6Z required ~20 min. (B) Emission spectra (ex: 640 nm)
demonstrating POPC@CSP⊃6Z was partially quenched compared with
POPC@CSP⊃6C.
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ZnDPA units in the newly docked 6Z with neighbouring CSP
conjugates, driven by Lewis acid/base interactions between the
Zn(II) cations and the electron rich squaraine oxygen atoms, as
reported for related metal cation/dye association systems.[39]
While the threading rate to form POPC@CSP
⊃
6Z liposomes was
slower than POPC@CSP 6C liposomes, it is important to
⊃
emphasize that both processes reached stoichiometric
completion in a practically useful time period. The second major
difference between POPC@CSP
liposomes was the fluorescence brightness. As shown in Figure
3B the fluorescence intensity of POPC@CSP 6Z liposomes was
about four times lower, which matches previous observations that
fluorescent ZnDPA complexes have a tendency to become self-
aggregated and self-quenched on a membrane surface.[28] As
shown below, relief of this self-quenching effect can be exploited
to achieve target-activated turn-on fluorescence.
6C and POPC@CSP 6Z
⊃ ⊃
Figure 4. (A) Photograph of three vials containing cationic beads treated with
(A1) buffer, (A2) POPC@CSP, and (A3) POPC@CSP 6C. (B) Fluorescent
⊃
micrographs (Ex: 635-675 nm, Em: 696-736 nm) showing an individual bead
from each of the corresponding samples in A. Red dashed line denotes bead
perimeter. Scale bar is 100 µm. (C) Fluorescence spectra (ex: 490 nm) of
⊃
supernatant from a sample of beads coated with POPC@CSP 6C liposomes
⊃
containing fluorescent rhodamine 123, before (red) and after (black) lysis of
the adhered liposomes with detergent.
The next stage of the research was to assess the targeting
a membrane lysis agent (Figure 4C). These results demonstrate
how Synthavidin self-assembly can be used to immobilize intact
fluorescent liposomes on synthetic surfaces for possible use in
nanotechnology applications such as sensing arrays.[40]
capabilities of the functionalized POPC@CSP
⊃
6C and
6Z liposomes. We expected that the anionic
6C liposomes would associate with cationic
POPC@CSP
POPC@CSP
⊃
⊃
surfaces, and this was confirmed by conducting a series of simple
experiments using rigid cationic polymeric beads (0.7 mm
diameter). As shown in Figure 4A-B, beads treated for 30 minutes
⊃
with POPC@CSP 6C liposomes, followed by extensive washing,
acquired a characteristic squaraine color and fluorescence, while
beads treated with untargeted POPC@CSP liposomes did not.
Evidence that the adhered POPC@CSP 6C liposomes were
⊃
structurally intact was gained by repeating the experiments using
liposomes containing rhodamine 123 (Rh) as a fluorescent
marker of aqueous contents. There was no escape of Rh from the
adhered liposomes until the sample was treated with detergent as
A more biomedically relevant set of targeting studies
⊃
examined cationic POPC@CSP 6Z liposomes with the
expectation they would selectively associate with anionic
membranes and cell surfaces. A series of model target liposomes
were prepared with membranes comprised of zwitterionic POPC
mixed with 0-20 mol% of anionic 1-palmitoyl-2-oleoyl-sn-glycero-
3-phosphoserine (POPS). Also incorporated into each of the
target membranes was 1 mol% of DiI which enabled membrane
targeting to be measured by FRET experiments (Figure 5A).[41]
Separate samples of functionalized and target liposomes were
Figure 5. (A) Association of POPC@CSP⊃6Z liposomes with anionic target liposomes containing POPS (20%) is indicated by FRET. (B) Change in FRET
with different target liposomes containing POPS (0-20%). (C) Dynamic light scattering showing size distributions at ten minutes after liposome mixing.
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Figure 6. Association of POPC@CSP⊃6Z liposomes (CSP was 3 mol% respective to POPC) with anionic target liposomes containing POPS (20%)
produces an increase in squaraine fluorescence (ex. 377 nm).
mixed and monitored for FRET from the DiI donor in the target
liposomes to the squaraine acceptor in the functionalized
⊃
liposomes. The results with POPC@CSP 6Z liposomes are in
Figure 5B and show that the extent of FRET increased with the
fraction of POPS in the target liposomes, and that the targeting
process reached completion after a few minutes. As expected,
there was no similar FRET increase when control experiments
lipid that is known to energetically favour a planar lamellar
structure and disfavour the curved surfaces that are formed during
the process of full membrane fusion,[42] and (b) the long PEG
chains that project from the membrane exterior hinder close
contact of the aggregated bilayers.[43,44] While full membrane
fusion and aqueous contents mixing is needed for effective
cytosol delivery of polar payload such as large macromolecules,
selective targeting of stable liposomes to cell surfaces is also a
desirable process for controlled delivery of lipophilic drugs[45] and
also for many imaging applications.[7]
were conducted using POPC@CSP
Each liposome mixture was also evaluated by DLS and only the
system that mixed POPC@CSP 6Z liposomes and POPS (20%)
liposomes produced a large increase in particle size (Figure 5C).
⊃
6C liposomes (Figure S12).
⊃
From a molecular recognition perspective, it is important to
It is worth noting that the aggregated liposomes did not precipitate, appreciate that the ZnDPA targeting units in POPC@CSP
⊃
6Z
most likely because the surrounding corona of PEG chains
provided the aggregates with colloidal stability. The liposome
aggregation process was further characterized by first measuring
liposomes are located near the liposome surface, at the base of
lengthy PEG-2000 chains. But the long PEG chains apparently do
not block the ZnDPA units from contacting the exposed surface of
target POPS (20%) liposomes. This finding encouraged us to
the amount of lipid mixing. Recalling that the embedded CSP
complex was self-quenched in the POPC@CSP 6Z liposomes,
a convenient lipid mixing assay was devised to measure the rate
that self-quenching was relieved. Thus, POPC@CSP 6Z
liposomes were prepared with ten times higher loading (3.0 mol%)
of the embedded CSP 6Z complex. Mixing these self-quenched
liposomes with target anionic POPS (20%) liposomes resulted in
a large increase in CSP 6Z fluorescence over a period of ~1
hour (Figure 6), indicating relief of self-quenching due to relatively
slow dilution of the embedded CSP 6Z into the associated POPS
⊃
6Z
⊃
determine if POPC@CSP 6Z liposomes could selectively target
⊃
a structurally more complicated biological cell surface and we
chose to test the clinically important bacterium Staphylococcus
aureus. We have previously shown that fluorescent ZnDPA
probes can selectively stain the anionic bacterial envelope in
preference to the near zwitterionic surfaces of healthy mammalian
cells.[46] Furthermore, multivalent ZnDPA probes are capable of
⊃
⊃
⊃
⊃
(20%) liposomes. Complementary information concerning
leakage of aqueous contents was gained by mixing
POPC@CSP
contained
⊃
a
6Z liposomes with POPS (20%) liposomes that
high concentration of the fluorescent dye,
sulforhodamine B (the chloride leakage assay used above is not
compatible with ZnDPA compounds). Self-quenching of the
sulforhodamine dye is relieved when it escapes the liposomes. As
shown in Figure S14 there was very little enhanced leakage from
the POPS (20%) liposomes after they were targeted by the
POPC@CSP
of leakage was observed when the sulforhodamine dye was
inside both the POPC@CSP 6Z liposomes and POPS (20%)
⊃
6Z liposomes. Furthermore, the same small level
⊃
liposomes (Figure S14), indicating negligible mixing of liposome
aqueous contents. Taken together, the data suggests that the
⊃
aggregated POPC@CSP 6Z and POPS (20%) liposomes are
not fully fused. That is, there is slow transfer of lipids between the
apposed bilayers but the aqueous interiors are not connected.
This conclusion is not surprising for a couple of reasons; (a) the
liposomes are primarily comprised of POPC, a cylindrical polar
Figure 7. Representative micrographs showing POPC@CSP⊃6Z liposomes
targeted and agglutinated S. aureus bacteria (B1,B2) but POPC@CSP
liposomes did not (A1,A2). Scale bar is 20 μm.
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Figure 8. Representative micrographs showing a mixture of bacteria (S. aureus) and human (Jurkat) cells treated with DiI (non-selective membrane dye)
and bacteria-selective POPC@CSP⊃6C liposomes. (A) Brightfield micrograph. (B) DiI fluorescence. (C) POPC@CSP⊃6C fluorescence. (D) Merge of the
three panels, with strong co-localization of DiI and POPC@CSP⊃6C indicated as yellow. Scale bar is 20 μm
strongly agglutinating bacteria in the presence of mammalian
cells.[46,47] With this precedence in mind we mixed separate
samples of planktonic S. aureus with either targeted
monolayer of liposomes and then threads a specific tetralactam
macrocycle onto the surface anchored CSP. The process
simultaneously equips the liposomes with targeting ligands,
sterically protective PEG chains, and deep-red fluorescence
reporter groups. Studies using a macrocycle bearing multiple
carboxylate groups produced anionic liposomes that adhered to
the surfaces of rigid cationic polymeric beads. Studies using a
macrocycle bearing ZnDPA targeting units produced
functionalized liposomes that recognized anionic membranes and
selectively agglutinated bacteria in the presence of mammalian
cells. It is notable that the corona of PEG chains around the
liposomes did not inhibit the targeting capabilities of the sheltered
ZnDPA targeting units. Using the same CSP conjugate as a
common docking platform, it should be possible to systematically
vary the macrocycle structure and create a wide range of targeted
liposomes for diagnostic or therapeutic applications.[50] Moreover,
Synthavidin self-assembly can be exploited to functionalize the
surface of many other types of biomedically important
nanoparticles,[51] especially those that are coated with bilayer
membranes such as exosomes,[14] microvesicles,[52] and cell-
membrane coated nanoparticles.[53]
POPC@CSP
⊃
6Z
liposomes
or
control
non-targeted
POPC@CSP liposomes. As shown by the representative
micrographs in Figure 7, the control liposomes had no
measurable affinity for the bacteria, whereas the targeted
liposomes produced immediate agglutination and co-localization
of the bacterial clusters with the liposome’s deep-red
fluorescence. Another set of cell microscopy experiments
assessed the ability of the POPC@CSP 6Z liposomes to target
⊃
the S. aureus bacteria in the presence of healthy human cells.
Separate samples of dispersed human cells (Jurkat) and S.
aureus were stained with a non-specific, fluorescent membrane
stain (DiI). Then the two cell populations were mixed and treated
with POPC@CSP 6Z liposomes. As shown by the brightfield
⊃
image in Figure 8, the bacterial cells were strongly agglutinated
and easily observed as clusters mixed with the much larger
human calls (additional images in Figures S15 and S16). As
expected, the exterior of both the human cells and the bacteria
were stained by the green-emitting DiI. In contrast, the deep-red
fluorescence of the POPC@CSP 6Z co-localized only with the
⊃
agglutinated bacteria. These microscopy studies clearly
demonstrate the selective targeting ability of the functionalized
⊃
POPC@CSP 6Z liposomes, and we infer that the targeting
Experimental Section
capabilities of the ZnDPA units located near the liposome surface
are not abrogated by the corona of long PEG chains that extend
above. This favourable outcome suggests that liposome
functionalization using Synthavidin self-assembly is a potentially
new way to solve the PEG dilemma,[9] which is the contradictory
vision of a liposome surface that is coated with targeting units and
protective PEG chains.[48,49]
Synthesis
The synthetic methods and compound characterization data are
described in the Supporting Information.
Liposome Experiments
General procedure:
Liposomes were prepared by the following sequence; a film of appropriate
phospholipid (1 μmol, purchased from Avanti Polar Lipids) was hydrated
with 1 mL of TES (5 mM pH 7.4) NaCl (145 mM), followed by dispersion
through brief vortex with a glass ring, five freeze/thaw cycles using liquid
N2 and a warm water bath, and extrusion 21 times through a 0.200 μM
polycarbonate filter.
Conclusions
Formation of POPC@CSP Liposomes:
A new self-assembly method is used to rapidly functionalize the
surface of liposomes without perturbing the membrane integrity or
causing leakage of the aqueous contents. The two-step method
first inserts the fluorescent conjugate CSP into the outer
An appropriate volume of CSP (50 μM) in water was added to a rapidly
stirred dispersion of pre-formed liposomes, to produce liposomes with
CSP (0.3 mol%, except where noted) only in the outer leaflet.
Alternatively, liposomes with CSP in both leaflets were prepared by
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including the CSP in the lipid film before hydration. Excitation spectra
with 720 nm emission and emission spectra with 640 nm excitation were
collected using 3 nm slits.
NaCl (145 mM). Fluorescence microscopy was conducted as before
using a Cy5.5-C filter set. Liposome leakage studies were conducted by
filling the liposomes with 30 µg/mL rhodamine 123 (Rh) and removing the
unincorporated Rh by size exclusion. The beads were treated with the
filled liposomes as before, washed 5 times, then added to the bottom of a
fluorescence cuvette with 1 mL of TES (5 mM pH 7.4), NaCl (145 mM)
supernatant, and a fluorescence scan of the supernatant was acquired
(ex. 490 nM). A small volume of Triton X-100 was then added to release
Rh into the supernatant, and another fluorescence scan was acquired.
Formation of POPC@CSP 6C and POPC@CSP 6Z Liposomes:
⊃ ⊃
Appropriate quantities of 6C or 6Z (100 μM) in water were added to
rapidly stirred dispersions of POPC@CSP liposomes. Excitation spectra
with 720 nm emission and emission spectra with 377 nm excitation
(anthracene absorption followed by internal energy transfer) were
collected using 3 nm slits and a 498 nm long pass filter in the emission
pathway to remove double diffracted scattered light.
FRET Experiments:
A chloroform solution of phospholipid and DiI (1 mol%) was evaporated
to create a film which was hydrated as described above. FRET from DiI
Acknowledgements
This work was supported by grants from the NSF (CHE1401783
to B.D.S.), the NIH (R01GM059078 to B.D.S. and
to CSP, CSP 6C, or CSP 6Z was monitored with ex: 545 nm, em: 560-
⊃ ⊃
800 nm, with 3 nm slit widths. A decrease in donor DiI emission and
increased emission at 690 nm (for the squaraine dye) or 710 nm (for the
threaded complex) was indicative of FRET.
T32GM075762 to S.K.S) and the CONACyT (Mexico).
Leakage Assays:
Keywords: liposomes • membranes • supramolecular chemistry
Chloride Leakage: POPC liposomes containing TES (5 mM, pH 7.4),
NaCl (145 mM) were prepared with an external solution of TES (5 mM),
NaNO3 (489 mM, pH 7.4). Chloride leakage into the external solution was
measured using a chloride selective electrode (Beckman) before and 30
min after addition of 0.3 mol% CSP and again after addition of 1.2 molar
equivalents of 6C. Calibration standards were made from isotonic
mixtures of the NaCl and NaNO3 buffers.
• fluorescent probes • cell recognition
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FULL PAPER
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10.1002/chem.201702649
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FULL PAPER
Insertion of a cholesterol-squaraine-
PEG conjugate into the outer leaflet of a
pre-formed liposome followed by
macrocycle threading of the squaraine
component produces a functionalized
deep-red fluorescent liposome.
Authors: Scott K. Shaw, Wenqi Liu,
Seamus P. Brennan, María de Lourdes
Betancourt-Mendiola, and Bradley D.
Smith
Non-Covalent Assembly Method that
Simultaneously Endows a Liposome
Surface with Targeting Ligands,
Protective PEG Chains, and Deep-Red
Fluorescence Reporter Groups
This article is protected by copyright. All rights reserved.