Capsosomes with "free-Floating" Liposomal Subcompartments

Article abstract of DOI:10.1002/adma.201100908

Capsosomes, that is, polymer capsules containing liposomal subcompartments, represent a promising new concept towards the design of artificial cells. Herein, capsosomes with control over the position of the subunits, "free-floating" in the capsule interior and/or associated with the polymer membrane, are reported. The functionality of these capsosomes is demonstrated via temperature-triggered encapsulated catalysis using subtilisin. Copyright

Full text of DOI:10.1002/adma.201100908

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Capsosomes with “Free-Floating” Liposomal
Subcompartments
Leticia Hosta-Rigau, Shiow Fong Chung, Almar Postma, Rona Chandrawati,
Brigitte Städler, and Frank Caruso*
Biological cells are able to perform multiple complex reactions
within confined environments, owing to their structures com-
prising internal subcompartments (e.g., cell organelles).[1] Arti-
ficial cells,[2–4] although far less complex than their biological
counterparts, can exhibit a hierarchical structure with a large
number of subcompartments confined within a structurally
stable scaffold. Such systems are engineered for therapeutic cell
mimicry via the encapsulation and/or conversion of biologically
active materials.[5]
poly(methacrylic acid) (PMA) carrier capsules, and preservation
of cargo functionality has been confirmed through enzymatic
assays[21,23] and cell viability studies.[24,25] In our previous work,
all of the capsosomes contain liposomal subcompartments
attached to the polymeric capsule wall. While the loading
efficiency of the subunits is not expected to be affected, the
functional activity of capsosomes could benefit from spatial
control over the positioning of the subcompartments, that is,
membrane-associated or “free-floating” liposomes in the cap-
sule interior (similar to the organelles in biological cells). This
is beneficial especially when fast mixing is essential or equal
access of reagents to the subcompartments is required, in par-
ticular when channel-gated liposomes are considered. Further,
controlling multistep reactions may be facilitated when the
subunits containing the reagent for the initial step are located
close to the scaffold membrane, while those loaded with rea-
gents for subsequent conversion and/or synthesis are situated
in the interior of the assembly.
With the goal to further advance our cell mimicry
approach, herein, we report the assembly of capsosomes
with controlled positioning of the liposomal subunits within
the polymer hydrogel carrier capsules via the pH-dependent
activity of hydrogen bonds between poly(N-vinyl pyrrolidone)
(PVP) and PMA (Scheme 1). In particular, we: i) synthesized
poly(N-vinyl pyrrolidone)-block-(cholesteryl acrylate) (PVPc),
a block copolymer consisting of a short block of cholesteryl
acrylate and a longer PVP segment, via reversible addition-
fragmentation chain transfer[26] (RAFT) and confirm the
selective adsorption of liposomes to PVPc; ii) demonstrate
the effect of sacrificial polymer layers on the loading effi-
ciency of liposomes into capsosomes; iii) confirm the pres-
ence of “free-floating” liposomes within capsosomes at
pH 7.4; iv) assembled capsosomes containing both “free-
floating” and membrane-associated liposomes; and v) demon-
strate preservation of cargo functionality by performing a
triggered enzymatic reaction encapsulating a protease in the
liposomal subcompartments.
Reported examples of subcompartmentalized micrometer-
sized assemblies include multiple subcompartments in the form
of micelles,[6] liposomes,[1,7–9] or polymersomes[10] entrapped
within a carrier vehicle; dual compartmentalized polymeric cap-
sules;[11,12] three-compartment microparticles;[13] polymer cap-
sules containing smaller polymer capsules,[14] cubosomes,[15]
or polymersomes;[16] capsules embedded in a polyelectrolyte
multilayer-stabilized gel bead;[17] and cellosomes, prepared by
the assembly of polyelectrolyte-coated yeast cells onto a tem-
plate that is subsequently removed.[18] Recently, we reported the
assembly of capsosomes–polymer (hydrogel) carrier capsules
containing liposomal subcompartments.[19] While the liposomes
provide protection for encapsulated fragile biological or small
hydrophobic and hydrophilic material, the polymeric capsule
provides the structural scaffold, and the semipermeable nature
of the polymer membrane enables interaction with the external
milieu. We have demonstrated that capsosomes are an efficient
functional encapsulation platform with potential use in drug
delivery or as artificial mimics of cells. In particular, we have
described capsosomes containing multilayers of different types
of liposomes (zwitterionic or negatively charged, saturated or
unsaturated) embedded within either nondegradable[20] or (bio)
degradable[21] polymer carrier capsules. Hydrophilic[21–23] and
hydrophobic[24,25] cargo have been encapsulated within the lipo-
somal subcompartments embedded within disulfide-stabilized
Dr. L. Hosta-Rigau, S. F. Chung, Dr. A. Postma, R. Chandrawati,
Dr. B. Städler,[+] Prof. F. Caruso
Department of Chemical and Biomolecular Engineering
The University of Melbourne
Parkville, Victoria 3010, Australia
E-mail: fcaruso@unimelb.edu.au
Electrostatic interactions alone can be limiting in the choice
of liposomes and polymers that can be assembled into a cap-
sosome due to restrictions in affinity between the liposomes
and the precursor and/or capping polymer layers. Hence, we
pioneered a non-covalent linkage concept based on the use of
cholesterol-modified polymers, which promotes successful
anchoring of the liposomes to a polymer film and does not
induce rupturing or displacement of the liposomes from the
surface upon deposition of the polymer capping layer.[21] We
previously introduced two cholesterol-containing polymers,
poly(methacrylic acid)-co-(cholesteryl methacrylate) (PMAc)
Dr. A. Postma
CSIRO Materials Science and Engineering
Clayton, Victoria 3168, Australia
[+] Present address: Interdisciplinary Nanoscience Center (iNANO),
Aarhus University, Aarhus 8000, Denmark
DOI: 10.1002/adma.201100908
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1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-
dioleoyl-sn-glycero-3-(phospho-L-serine)(DOPS)(DOPC:DOPS=
4:1 wt/wt, Lu,−), and fluorescent lipids (1 wt% 1-myristoyl-
2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]-hexanoyl]-sn-
glycero-3-phosphocholine (NBD-PC)), were employed to maxi-
mize the electrostatic repulsion between the negatively charged
disulfide-stabilized PMA carrier capsule and the liposomes at
physiological conditions. The carboxylic groups of the PMA
chains are deprotonated at pH 7.4, and this should facilitate
detachment of the liposomes from the polymer membrane.
The fluorescence intensity of silica particles due to adsorp-
tion of fluorescently labeled liposomes was monitored by flow
cytometry (Figure 1a). The largest amount of liposomes depos-
ited was observed when the liposomes were adsorbed onto
silica particles only precoated with PVPc. The adsorption of
PVP/PMA bilayers prior to PVPc reduced the amount of lipo-
somes adsorbed by at least 70%. This observation is attributed
to a reduced number of cholesteryl units protruding from the
surface due to the presence of underlying polymer bilayers and,
hence, less units being accessible for anchoring the liposomes.
Importantly, there was negligible adsorption of liposomes to
the particles coated with unmodified PVP as a precursor layer,
Scheme 1. Schematic illustration of a capsosome containing “free-
floating” liposomal subcompartments. The first inset shows the block
copolymer PVPc coating a liposome (i), while the second inset is an illus-
tration of the intercalation of the cholesteryl moieties attached to the PVPc
into the lipid bilayer of a liposome (ii).
and cholesterol-modified poly(L-lysine) (PLLc).[21] Both of these
polymers lead to the formation of capsosomes but neither
of them promotes the formation of “free-
floating” liposomes in the capsule interior.
This is not surprising for PLLc because of its
electrostatic attraction with the PMA of the
carrier capsule. PMAc, on the other hand, is
negatively charged and is not cross-linked to
the polymer membrane of the carrier, but the
liposomes remain associated with the capsule
wall, most likely due to physical entanglement
of the copolymer with the building blocks
of the polymer membrane. We therefore
utilize a tailor-made cholesterol-containing
block-copolymer that is (a) uncharged and
(b) contains cholesterol moieties in a short
block to minimize the entanglement of
the liposome-anchoring polymer with the
PMA capsule; namely PVPc. This polymer
can anchor liposomes via its cholesteryl-
block while the uncharged PVP-block forms
hydrogen bonds with PMA only during the
assembly of the carrier membrane (pH 4) but
not at physiological conditions (pH 7.4).
The RAFT synthesized block copolymer,
PVPc, was found to have a total molecular
weight of 10.9 kDa, with 3 mol% of choles-
terol and a narrow polydispersity of 1.22
(for more details see the Supporting Infor-
mation, Scheme S1, Figure S1 and S2). We
examined the ability of PVPc to adsorb lipo-
somes onto 3-μm-diameter silica particles
via noncovalent anchoring of the cholesterol
moieties to the lipid membrane. Addition-
ally, we compared the amount of adsorbed
liposomes onto particles with different
numbers of preadsorbed bilayers of PVP
and PMA. Negatively charged fluorescently
Figure 1. a) Fluorescence intensity of silica particles pre-coated with a variable number of
PVP/PMA bilayers and PVPc or PVP upon the adsorption of fluorescently labeled liposomes
(Lu,−NBD). b) The fluorescence intensity of particles or capsosomes due to the presence of
Lu,−NBD at different stages of the assembly: after the initial liposome deposition (black), after the
assembly of the core-shell particles (dark grey), capsosomes in NaOAc buffer (light grey) and
in HEPES buffer (striated light grey). Red arrows indicate the decrease in fluorescence intensity
of the capsosomes upon buffer change. c) CLSM images of capsosomes (PVPc/Lu,−NBD/PVPc/
PMA/PVP/(PMASH/PVP)4) showing the liposomes associated with the polymer membrane in
NaOAc buffer (i) and “free-floating” within the capsules in HEPES buffer (ii). In HEPES buffer,
labeled unsaturated liposomes (Lu,−NBD), the PMASH membrane has been post-labeled with Alexa Fluor-633 maleimide.
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demonstrating the need for the cholesteryl moieties of PVPc to
anchor the liposomes.
both conditions. Confocal laser scanning microscopy (CLSM)
images of PVPc/Lu,−NBD/PVPc/PMA/PVP/(PMASH/PVP)4 in
NaOAc buffer showed regular green fluorescence associated
with the capsosome membrane, which arose from the green
labeled liposomes (Figure 1c(i)). Upon buffer change, the
hydrogen bonds between the PVPc and PMA are disrupted
and thus the liposomes trapped between the PVPc layers were
detached from the membrane of the carrier capsule. This is
shown by the green fluorescence homogeneously distributed
within the capsosomes (Figure 1c(ii)). The “free-floating” green
liposomes were stably entrapped within the red-labeled PMA
hydrogel carrier capsules (the polymer membrane was post-
labeled with (Alexa Fluor-633)-maleimide in HEPES buffer).
Further, detachment of the liposomes was found to be revers-
ible. Upon reverting back to NaOAc buffer, the liposomes reat-
tached themselves to the polymer membrane, suggesting that
the PVPc was not released from the capsosomes, but rather that
the cholesterol-modified polymer remained largely associated
with the liposomes (Supporting Information, Figure S5). From
fluorescence analysis, we estimate that each 3-μm-diamater
capsosome contains ≈5000 “free-floating” liposomal subcom-
partments (see the Supporting Information).
After having identified PVPc as a specific and suitable pre-
cursor polymer layer for Lu,−
adsorption (without the need
NBD
for PVP/PMA bilayers), capsosomes were assembled by cap-
ping the liposomes with PVPc and subsequently adsorbing a
variable number of PMA/PVP separation/sacrificial bilayers.
These polymer bilayers are expected to serve as sacrificial
layers because they disassemble under specific conditions, con-
sequently assisting in detachment of the liposomes from the
polymer membrane. The polymer carrier capsules were subse-
quently created by the sequential deposition of four bilayers of
thiol-modified PMA (PMASH) and PVP in sodium acetate buffer
(20 mM NaOAc, pH 4).[27] The thiol groups in the polymer
multilayer film of these core–shell particles were cross-linked
into disulfide linkages using chloramine T prior to dissolution
of the template particles in buffered hydrofluoric acid (HF) to
yield capsosomes. The retention efficiency of the fluorescently
labeled liposomes within the polymer multilayer film as a func-
tion of the number of sacrificial separation bilayers was assessed
by flow cytometry. The fluorescence intensity of the particles
due to the initial adsorption of fluorescent liposomes was set to
100% and compared to the fluorescence intensity of the core–
shell particles after cross-linking as well as the capsosomes (after
core dissolution) in NaOAc buffer and in HEPES buffer [10 mM
HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid),
150 mM NaCl, pH 7.4] (Figure 1b). The core–shell particles and
the capsosomes in NaOAc buffer showed similar fluorescence
intensities for all of the different assemblies, suggesting that the
different number of separation PMA/PVP bilayers did not affect
liposome retention. However, upon changing the environment
of the capsosomes from NaOAc to HEPES buffer, the number of
stably trapped liposomes varied, depending on the number of
separation PMA/PVP bilayers. Increasing the number of sacri-
ficial separation bilayers caused a decrease of the fluorescence
intensity of the capsosomes upon buffer change (Figure 1b).
While in the absence of PMA/PVP bilayers a reduction of only
≈10% was observed, the presence of two PMA/PVP bilayers gave
rise to ≈45% lower intensity. The increase in pH results in the
disruption of the hydrogen bonds, and thus the (sacrificial) PVP
and nonthiolated PMA were destabilized and released, possibly
either dragging the liposomes along or temporarily increasing
the permeability of the polymer membrane, which might facili-
tate loss of the liposomes. Release of PVP from the multilayer
film has previously been shown,[28] and the release of PMA at
pH 7.4 was confirmed (Supporting Information, Figure S3).
The yellow signal arising from the superposition of green fluo-
rescently labeled liposomes (Lu,−NBD) and red PMAAF633 was
observed (Supporting Information, Figure S3a). Upon buffer
change, the sacrificial layers were disrupted and PMAAF633 was
released (Supporting Information, Figure S3b). Capsosomes
were assembled via this process (i.e., using PVPc as precursor
and capping layers without additional PMA/PVP sacrificial
bilayers) with the goal to achieve the highest liposome reten-
tion and were characterized both in NaOAc buffer and under
physiological conditions using optical and fluorescence micro-
scopy. Differential interference contrast images (Supporting
Information, Figure S4) demonstrated that the capsosomes
obtained were structurally intact and non-aggregated under
We next sought to obtain capsosomes comprising both “free-
floating” and membrane-associated liposomes. We assembled
liposomes in two deposition steps, expecting the inner lipo-
some layer to become “free-floating” while the outer layer to
remain associated with the polymer membrane. The first layer
of liposomes was adsorbed onto PVPc pre-coated silica par-
ticles while the second layer was anchored to (PVPc/Lu,−
/
NBD
PVPc/PMA/PVP/PMAc)-modified particles. The measured
fluorescence intensity of the silica particles after the first and
the second liposome deposition steps showed that a similar
number of liposomes adsorb in each step (Supporting Infor-
mation, Figure S6), demonstrating that the initial layers did
not hinder subsequent liposome assembly. Capsosomes were
then formed by capping with PMAc and assembling the PVP/
PMASH polymer membrane, followed by cross-linking of the
thiols in the film and removing the core. CLSM images of these
capsosomes containing NBD-labeled (Lu,−NBD) and rhodamine-
labeled (Lu,−Rhd) liposomes consisting of DOPC:DOPS = 4:1
wt/wt and fluorescent lipids 1 wt% 1,2-dipalmitoyl-sn-glycero-
3-phosphoethanolamine-N-(lissamine Rhodamine B sulfonyl)
(Rhd-PE) showed both types of liposomes associated with the
polymer membrane in NaOAc buffer (assembly conditions)
(Figure 2a(i) and 2a(i′). Upon changing the buffer to HEPES,
the green fluorescently labeled liposomes were observed to be
“free-floating” inside the capsosomes, as shown by the homo-
geneously distributed green emission (Figure 2a(ii)). The red-
fluorescently labeled liposomes remained attached to the shell
(Figure 2a(ii′)), as shown by the red emission emerging from the
walls of the capsules. This demonstrates spatial control over the
positioning of the liposomal subcompartments, a crucial step
forward in assembling confined hierarchical structures. How-
ever, when assembling the same system but containing unla-
belled liposomes in the inner layer (Lu,−) and green labeled lipo-
somes in the outer layer (Lu,−NBD), the green labeled liposomes
became “free-floating” in the capsule interior in HEPES buffer
(Supporting Information, Figure S7). These results suggest that
control over the position of the liposomes is a combination of
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(i) the different properties of PVPc and PMAc and (ii) the dif-
ferent fluorophore-conjugated lipids (i.e., NBD-PC and Rhd-
PE). For the former case, the assembly with a PMAc capping
layer resulted in membrane-associated liposomes (Figure 2b(i)),
while when assembling the same capsosomes but using PVPc
as a capping layer, the liposomes became “free-floating” at phys-
iological conditions (Figure 2b(ii)). For the latter case, the lipid
NBD-PC contains the fluorophore attached to the hydrophobic
tail of the phospholipids (Supporting Information, Figure S8a),
while the Rhd-PE lipid contains the fluorophore anchored to
the hydrophilic head of the phospholipid (Supporting Infor-
mation, Figure S8b). Thus, when assembling liposomes con-
taining NBD-PC lipids, the fluorophore will be enclosed within
the lipid membrane of the liposomes and therefore is unlikely
to interact with other compounds, while the fluorophore of the
Rhd-PE lipids will remain exposed for interaction with other
species.
To examine the functionality of capsosomes containing “free-
floating” liposomes, we encapsulated the protease subtilisin in
the subunits of the capsosomes and performed a triggered enzy-
matic reaction by converting the coumarine peptidase substrate
N-CBZ-L-phenylalanyl-L-arginine 7-amido-4-methylcoumarin
(AMCs) into its hydrolyzed product 7-amino-4-methylcoumarin
(AMCp). Depending on the purpose of the capsosomes, different
types of liposomes might be employed. For the encapsulation
of enzymatic cargo, saturated liposomes are preferably used
due to their higher phase-transition temperature (Tm). Nega-
tively charged saturated liposomes (Ls,−NBD), 1,2-dimyristoyl-sn-
glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-
3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-
(phospho-L-serine) (DPPS) (DMPC:DPPC:DPPS = 8:1:1 wt/
wt, Tm ≈ 28 °C) and fluorescent lipids (1 wt% of NBD-PC)
were employed to assemble PVPc/Ls,−NBD/PVPc/PMA/PVP/
(PMASH/PVP)4, and after cross-linking and core removal capso-
somes were observed by fluorescence microscopy. As depicted
in Figure 2c(i), in NaOAc buffer a regular green fluorescence
signal arising from the shell of the capsosomes was observed,
while upon changing to HEPES buffer, the green fluorescence
signal was homogeneously distributed throughout the capso-
some cavity (Figure 2c(ii)). This demonstrates “free-floating”
saturated liposomes in the capsule interior. The choice of the
enzyme subtilisin and the AMCs made it possible to follow
and quantify the conversion of the substrate into the product
by fluorescence spectrophotometry (AMCp excitation/emission
maxima at ≈351/430 nm, Supporting Information, Figure S9).
Saturated negatively charged liposomes loaded with subtilisin
(Ls,−Sub) were assembled in two different ways, one consisting
of PVPc/Ls,−Sub/PVPc/PMA/PVP/(PMASH/PVP)4-capsosomes,
which promotes “free-floating” of the liposomal subcompart-
ments, andPLL/Ls,−Sub/PMAc/PVP/(PMASH/PVP)4-capsosomes,
which generates capsosomes containing liposomes attached
to the inside of the polymer shell (Figure 3a). We used the
enhanced permeability of the liposomal membrane at elevated
temperature to trigger the enzymatic reaction. Equal numbers
of subtilisin-containing capsosomes were incubated with AMCs
and heated at T = 23 °C (RT, room temperature) or at T = 37 °C
(above the Tm of the liposomes). AMCs was also incubated with
free subtilisin at T = 23 °C (Figure 3b, column 1) or at T = 37 °C
(Figure 3b, column 2) at a similar concentration as the subtilisin
Figure 2. a) CLSM images of capsosomes (PVPc/Lu,−NBD/PVPc/PMA/
PVP/PMAc/Lu,−Rhd/PMAc/PVP/(PMASH/PVP)4) containing
a layer of
green (i), (ii) and red (i′), (ii′) labeled liposomes in NaOAc buffer and in
HEPES buffer (left and right, respectively). b) Fluorescence microscopy
images of (i) PVP/PMAc/Lu,−NBD/PMAc/PVP/PMA/PVP/(PMASH/PVP)4-
capsosomes and (ii) PVPc/Lu,−NBD/PVPc/PMA/PVP/(PMASH/PVP)4-
capsosomes at physiological conditions. c) Fluorescence microscopy
images of PVPc/Ls,−NBD/PVPc/PMA/PVP/(PMASH/PVP)4-capsosomes in
(i) NaOAc buffer and (ii) in HEPES buffer.
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by the free subtilisin in solution (≈90%) (Figure 3b, dark grey
columns 4, 6, and 2). Upon increasing the temperature the
liposomal membrane permeability is enhanced, and the small
substrate AMCs is able to cross through the liposomes, thus
interacting with the enzymes and being converted into AMCp
(Figure 3a). This result demonstrates the efficiency in the
encapsulation of the enzyme within the liposomal subcompart-
ments and preservation of cargo functionality (we achieved a
similar conversion of AMCs when the enzyme is trapped in the
liposomal subcompartments and when the enzyme is free in
solution). Although no difference was observed between capso-
somes containing “free-floating” or membrane-associated sub-
compartments when incubated at T = 37 °C, the control sam-
ples left at RT showed a difference. After 24 h it was observed
that capsosomes containing membrane-associated liposomes
showed ≈10% conversion of the substrate (Figure 3b, column 3,
dark grey) while the “free-floating” liposomes converted ≈50%
of the substrate into its fluorescent product (Figure 3b, column
5, dark grey). This confirms that temperature can be used to
trigger the enzymatic reaction. In addition, this result gives
rise to the possibility of using capsosomes with “free-floating”
subunits in a continuous manner, that is, without the need of a
trigger to initiate the reaction. The differences between the two
types of capsosomes can be explained due to the different poly-
mers used for their assembly. When assembling capsosomes
containing the liposomal subcompartments attached to the
shell walls we used PMAc, a graft copolymer. In contrast, when
assembling capsosomes containing “free-floating” subcompart-
ments, a block copolymer PVPc was used. The different distri-
bution of the cholesteryl pendant units along the polymer chain
as well as the type of polymer is likely to affect the liposomal
membrane and is currently part of a more detailed investiga-
tion. Further, we incubated the same capsosomes with AMCs
solution one week after the assembly and performed the same
enzymatic assay with the goal to demonstrate preservation of
enzyme activity since the proteases are prone to self-degradation.
As depicted in Figure S10 (Supporting Information), the enzy-
matic activity of subtilisin is preserved when it is stored in
solution or encapsulated in capsosomes containing either
“free-floating” or membrane-associated liposomes.
In conclusion, the synthesis and use of a novel block copolymer
PVPc to anchor liposomes to a polymer film allows control over the
spatial positioning of liposomes entrapped within a PMA hydrogel
carrier capsule. We demonstrated that with the appropriate use of
cholesterol-modified polymers and PMA/PVP sacrificial layers,
capsosomes with “free-floating” and polymer membrane-
associated subcompartments can be prepared. The controlled
enzymatic conversion of AMCs into its hydrolyzed product dem-
onstrates the preservation of cargo functionality when the enzyme
subtilisin is encapsulated into the “free-floating” or membrane-
associated liposomal compartments of capsosomes. These are
pivotal features on the way to further developing capsosomes into
a functional biomedical platform and as mimics for cells.
Figure 3. a) Schematic illustration of the enzymatic conversion of AMCs
into AMCp using capsosomes loaded with the protease subtilisin upon
increasing the temperature above Tm. b) Fluorescence intensity of the
AMCp after 4, 17, and 24 h due to the enzymatic activity by: free subtilisin
at a similar concentration as the subtilisin entrapped within the liposomal
compartments of the capsosomes incubated at 23 °C (1) or 37 °C (2);
capsosomes containing the subtilisin-loaded membrane-associated lipo-
somal compartments at 23 °C (3) or 37 °C (4); and capsosomes con-
taining the subtilisin-loaded “free-floating” liposomal compartments
incubated at 23 °C (5) or 37 °C (6).
encapsulated in the capsosomes. As depicted in Figure 3b, the
fluorescence intensity of AMCp was measured at 436 nm after
4 h (white columns), 17 h (light grey columns) and 24 h (dark
grey columns) of incubation. After 24 h of incubation, both cap-
sosomes containing liposomes attached to the polymer shell
and capsosomes containing “free-floating” subcompartments at
37 °C showed a normalized fluorescence intensity of ≈100%, a
value close to the fluorescence intensity of the AMCp converted
Experimental Section
Capsosomes with “free-floating” liposomal subcompartments were
assembled via the layer-by-layer technique. A suspension of 3.25-μm-
diameter silica particles (5 wt%) in NaOAc buffer was incubated with
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PVPc (1 mg mL−1 , 15 min), washed three times, suspended in the
liposome solution (1.25 mg mL−1 , 40 min), and washed again three times,
followed by incubation with PVPc (1 mg mL−1 , 15 min). A sacrificial layer
PMA (1 mg mL−1 , 10 min) was adsorbed and the polymer membrane
of the carrier capsules was assembled by sequential deposition of four
bilayers of PVP and PMASH (1 mg mL−1 , 10 min). The thiols within the
polymer layers were cross-linked with 1,8-bismaleimidodiethyleneglycol
(BM(PEG)2) (2 mM, 15 h) in MES buffer (50 mM MES (2-(N-morpholino)-
ethanesulfonic acid), pH 6.0), and the cores were removed using a
2 M HF/8 M NH4F solution for 2 min, followed by several washing cycles
in NaOAc buffer (4500 g, 3 min). A CyFlow Space (Partec Flowmax,
Münster, Germany) flow cytometer using an excitation wavelength of
488 nm was used for all of the flow cytometry experiments. The
fluorescently labeled capsosomes were imaged using a Leica TCS SP2 SE
confocal microscope. The conversion of the AMCs into its product was
monitored over time by fluorescence spectrophotometry (Fluorolog-3
Model FL3-22 spectrofluorometer) using an excitation wavelength of
351 nm and recording the fluorescence intensity at 436 nm.
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Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
Acknowledgements
This work was supported by the Australian Research Council under
the Federation Fellowship (F.C.) and Discovery Project (F.C.) schemes.
We thank Dr. Alexander N. Zelikin (Chemistry Department, Aarhus
University, Denmark) and Siow-Feng Chong (The University of
Melbourne, Australia) for their input in assembling the PMA hydrogel
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Received: March 10, 2011
Published online: July 27, 2011
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