Polymer-caged lipsomes: A pH-responsive delivery system with high stability

Article abstract of DOI:10.1021/ja070748i

Polymer-incorporated liposomes were prepared from preformed liposomes and a cholesterol-functionalized poly(acrylic acid) additive via a simple drop-in procedure. These modified liposomes possess surface-active carboxylate groups that can be cross-linked with telechelic 2,2′-(ethylenedioxy)bis(ethylamine) linkers, resulting in polymer-caged liposomes (PCLs) that are highly stable and have tunable pH-sensitive responses. Copyright

Full text of DOI:10.1021/ja070748i

Published on Web 11/14/2007
Polymer-Caged Lipsomes: A pH-Responsive Delivery System with High
Stability
Sang-Min Lee, Haimei Chen, Christine M. Dettmer, Thomas V. O’Halloran,* and SonBinh T. Nguyen*
Department of Chemistry and the Center for Cancer Nanotechnology Excellence, Northwestern UniVersity,
EVanston, Illinois 60208-3113
Received February 1, 2007; E-mail: t-ohalloran@northwestern.edu; stn@northwestern.edu
Scheme 1. Synthesis of Polymer-Caged Liposomes (PCLs)
Liposomes are self-assembled vesicles consisting of a spherical
bilayer structure surrounding an aqueous core domain. Due to their
intrinsic biocompatibility and ease of preparation, several liposomal
drugs have been approved.1 In addition, modified liposomes on the
nanoscale (∼120 nm) have been shown to have excellent pharma-
cokinetic profiles2 for the delivery of nucleic acids, proteins, and
chemotherapeutic agents such as doxorubicin. However, major
drawbacks of liposome-based drug carriers include their instability
and the lack of tunable triggers for drug release. As such, there
have been several attempts at enhancing the properties of lipo-
somes.1,3 Incorporation of polymerizable lipid amphiphiles leads
to cross-linked liposomes with higher stability.4 Unfortunately, every
lipid system would require a specific polymerizable amphiphile,
making this approach synthetically cumbersome. In addition, the
cross-links are often too stable to allow for controllable releases of
the payload. To provide a combination of stability and modification
generality, hydrophilic polymers such as poly(ethylene glycol)
(PEG)2 and poly(N-isopropylacrylamide)5 have been added to
liposomes. However, these modifiers can easily dissociate from the
liposome surface, returning them to the unstable state.6 Herein, we
demonstrate a general drop-in strategy that allows for long-term
stabilization of virtually any liposome system via a biocompatible
polymer cage.
Cross-linking of surface functional groups in self-assembled
polymer nanoparticles has been shown to prevent the dissociation
of polymer components.7 In addition, highly functionalized polymer
nanomaterials can be engineered to change shape via external
stimuli such as pH and temperature.8 Our work combines both of
these design features to arrive at a single polymer amphiphile that
can be used to stabilize any liposome system while allowing for
additional attributes such as tunable drug-releasing properties and
targeting ability. In particular, we demonstrate that a cholesterol-
terminated poly(acrylic acid) (Chol-PAA) can be readily inserted
into a known liposome system and then cross-linked to stabilize
the bilayer membrane (Scheme 1). The resulting polymer-caged
liposomes (PCLs) are highly stablesthey can be lyophilyzed into
powder forms and redispersed without loss of structural coherence.
They can also be induced to release a model payload (calcein) under
acidic conditions. As such, they may have applications as environ-
ment-specific nanoscale delivery vehicles.
dioleoylphosphatidylglycerol, and cholesterol with molar ratio of
51.4:3.6:45, Figure 1A) and incubated overnight to yield polymer-
incorporated liposomes11 (PILs, Figure 1B). After the incubation,
only particles with increased mean DH (93 ( 15 nm, PDI ) 0.047
( 0.016) was observed, suggesting the homogeneous formation of
PILs (Figure S4). Cross-linking of the poly(acrylic acid) moieties
on the surface of PILs was achieved using 2,2′-(ethylenedioxy)-
bis(ethylamine), and the formation of amide bond in the resulting
1
PCLs (Figure 1C) was observed by water-suppressed H NMR
spectra (Figure S7). The apparent hydrodynamic diameter of the
polymer-shell increased (Figure S9), indicating that significant
cross-linking has occurred.
Our PCLs are remarkably stablestheir spherical structures were
fully preserved after freeze-drying and rehydration (Figure 2A). In
contrast, the same treatment completely destroyed the spherical BLs
(Figure S6), presumably due to loss of the aqueous core as well as
increased surface-surface interactions that allow for establishment
of broad lamellae structures.12 The outstanding stability of our PCLs
under the lyophilization/rehydration process suggests that they can
be stored on a long-term basis, a desirable feature in delivery
applications.
For in vivo applications, drug carrier vesicles must possess high
plasma stability. Accordingly, we evaluate the stability of a calcein-
encapsulated sample of our PCLs against fetal bovine serum (FBS)
at 37 °C following a procedure described by Allen et al.13 As
calcein’s fluorescence is self-quenched under the high concentra-
tions found in intact liposomes, its leakage due to vesicle rupture
is readily observed. Remarkably, only a minute leakage of calcein
(∼5%) was observed from our PCLs after 500 h, an order of
magnitude less than the leakage in BLs (Figure 2B). We attributed
the inhibition of PCL rupture to the steric barrier provided by the
cross-linked polymer shell. Although incorporation of PEG-
conjugated phospholipids into liposomes has been reported to
sterically stabilize the resulting vesicles,2 long-term stability was
still low due to the rapid dissociation of the water-soluble polymer-
attached lipids from the bilayer membrane.6b In a similar manner,
Narrowly dispersed cholesterol-terminated poly(acrylic acid) (Mn
) 2.5 kDa, Mw/Mn ) 1.1) was synthesized via nitroxide-mediated
controlled radical polymerization9 of tert-butyl acrylate followed
by acidolysis (Figures S1-S3 in Supporting Information (SI)). We
employed poly(acrylic acid) as a hydrophilic polymer due to its
biocompatibility and easily cross-linkable carboxylate group. The
cholesterol end group acted as a single anchor to eliminate the
possible aggregation often seen with polymers including multi-
anchor groups.10 Chol-PAA (10 mol % compared to the total
amount of lipids) was mixed with a solution of bare liposomes (BLs,
DH ) 82 ( 14 nm, PDI ) 0.046 ( 0.021 via dynamic light
scattering (DLS), prepared from dipalmitoyl-phosphatidylcholine,
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10.1021/ja070748i CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
the membrane, decreasing lipid-lipid interactions responsible for
membrane stabilization.14 Both of these effects can lead to a collapse
of the cross-linked shell and a subsequent compression/rupture of
the PCL core, mechanically similar to that observed for a cross-
linked poly(acrylamide-acrylic acid) hydrogel.15 These effects
perturb the membrane structure and may induce the formation of
pores that are sufficiently large to allow for the leakage of the
calcein contents.16
In summary, polymer-caged liposomes were readily prepared
from preformed liposomes and a cholesterol-functionalized poly-
(acrylic acid) additive via a facile drop-in procedure. The highly
enhanced stability and tunable pH-sensitive responses of these novel
materials are made possible entirely by the environmental responsive
properties of the encapsulating polymer shell. Our simple but highly
effective strategy could be used to modify many clinically relevant
liposome-based drug-delivery systems, including inorganic drug-
encapsulated liposomes.17 In addition, as the cross-linked polymer
shell still possess unmodified carboxylic acid groups, it can be
further functionalized with antibody- and ligand-based targeting
groups using “post-particle-formation modification” strategies.18 The
results from such studies will be reported in due course.
Figure 1. Transmission electron microscope (TEM) images of (A) bare
liposomes (BLs), (B) polymer-incorporated liposomes (PILs), and (C)
polymer-caged liposomes (PCLs). All samples were negatively stained with
4% uranyl acetate. Both wholly spherical (A) and indented spherical
morphologies (B and C) are commonly observed in liposomal TEM (see
SI).
Acknowledgment. Financial support by the NIH (NCI Center
for Cancer Nanotechnology Excellence U54CA119341 and Core
Grant P30CA060553 to the Northwestern Lurie Cancer Center) and
the NSF (DMR-0094347 and EEC-0647560) is appreciated. We
acknowledge the use of instruments in the Northwestern NUANCE
and ASL facilities (see SI for funding information).
Supporting Information Available: Synthetic and preparative
procedures and characterization data for Chol-PAA, BL, PIL, and PCL.
This material is available free of charge via the Internet at http://
pubs.acs.org.
Figure 2. (A) TEM image of PCLs after freeze-drying and rehydration.
(B) Calcein leakage assay of BLs, PILs, and PCLs at 37 °C in fetal bovine
serum (FBS). (C) Acid-triggered calcein release at 37 °C and (D) temporal
evolution of mean DH in pH 7.4 and 5.5.
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