Polymersomes prepared from thermoresponsive fluorescent protein-polymer bioconjugates: Capture of and report on drug and protein payloads

Article abstract of DOI:10.1002/anie.201412406

Polymersomes provide a good platform for targeted drug delivery and the creation of complex (bio)catalytically active systems for research in synthetic biology. To realize these applications requires both spatial control over the encapsulation components in these polymersomes and a means to report where the components are in the polymersomes. To address these twin challenges, we synthesized the protein-polymer bioconjugate PNIPAM-b-amilFP497 composed of thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) and a green-fluorescent protein variant (amilFP497). Above 37 °C, this bioconjugate forms polymersomes that can (co-)encapsulate the fluorescent drug doxorubicin and the fluorescent light-harvesting protein phycoerythrin 545 (PE545). Using fluorescence lifetime imaging microscopy and F?rster resonance energy transfer (FLIM-FRET), we can distinguish the co-encapsulated PE545 protein inside the polymersome membrane while doxorubicin is found both in the polymersome core and membrane.

Full text of DOI:10.1002/anie.201412406

Angewandte
Chemie
DOI: 10.1002/anie.201412406
Polymersomes
Hot Paper
Polymersomes Prepared from Thermoresponsive Fluorescent Protein–
Polymer Bioconjugates: Capture of and Report on Drug and Protein
Payloads**
Chin Ken Wong, Alistair J. Laos, Alexander H. Soeriyadi, Jçrg Wiedenmann, Paul M. G. Curmi,
J. Justin Gooding, Christopher P. Marquis, Martina H. Stenzel, and Pall Thordarson*
Abstract: Polymersomes provide a good platform for targeted
drug delivery and the creation of complex (bio)catalytically
active systems for research in synthetic biology. To realize these
applications requires both spatial control over the encapsula-
tion components in these polymersomes and a means to report
where the components are in the polymersomes. To address
these twin challenges, we synthesized the protein–polymer
bioconjugate PNIPAM-b-amilFP497 composed of thermores-
ponsive poly(N-isopropylacrylamide) (PNIPAM) and
I
nspired by nature, where compartmentalization of biolog-
ical components is all pervasive, we are interested in
developing a platform technology of self-assembled multi-
functional compartments with applications in areas ranging
from nanomedicine[1–4] to synthetic biology.[1,2,5–8] Vesicles and
polymersomes that either contain entrapped biomole-
cules[9–11] or are made from protein–polymer biohybrids[12–14]
are the archetypical example of such multifunctional systems.
Due to their enhanced stability when compared to liposomes,
polymersomes are particularly attractive for applications such
as targeted drug delivery.[2,4,15–16] For similar reasons they also
provide a good platform for compartmentalizing multien-
zyme cascades[10] and other sophisticated systems related to
synthetic biology.[17,18] There are, however, a number of
challenges with polymersomes. These challenges include:
1) the formation and morphology is not easily controlled,
making methods to initiate the formation of well-defined
polymersomes highly valuable, 2) designing a generic poly-
mersome system that can encapsulate a payload ranging from
drugs to proteins at precise locations in the polymersomes,
and 3) a means of monitoring the location and status of the
payload of interest within the polymersome.
a
green-fluorescent protein variant (amilFP497). Above
378C, this bioconjugate forms polymersomes that can (co-
)encapsulate the fluorescent drug doxorubicin and the fluo-
rescent light-harvesting protein phycoerythrin 545 (PE545).
Using fluorescence lifetime imaging microscopy and Fçrster
resonance energy transfer (FLIM-FRET), we can distinguish
the co-encapsulated PE545 protein inside the polymersome
membrane while doxorubicin is found both in the polymer-
some core and membrane.
[*] C. K. Wong, A. J. Laos, Dr. A. H. Soeriyadi, Prof. J. J. Gooding,
Assoc. Prof. P. Thordarson
Herein, we report the preparation of well-defined, multi-
functional, biohybrid polymersomes that responds to all these
challenges. The polymersomes are formed by the self-
assembly of fluorescent protein–thermoresponsive polymer
bioconjugates (Scheme 1). The design of the polymer com-
ponent is crucial to the final polymersome function and the
versatility of the system. A narrow polydispersity of the
polymer–protein conjugate ensures that well-defined low-
polydispersed polymersomes are formed. The thermorespon-
sive polymer component, ideally around 378C, corresponding
to human body temperature, provides a means to form and
dissociate the polymersomes so drugs and proteins can be
loaded and released from the polymersomes by simply
changing the temperature. Further, the fluorescence property
of the protein component allows a combination of fluores-
cence lifetime imaging microscopy (FLIM) and Fçrster
resonance energy transfer (FRET; FLIM-FRET[19–21]) which
precisely reveals the location of the encapsulate within the
polymersome.
School of Chemistry, The Australian Centre for NanoMedicine and
the ARC Centre of Excellence in Convergent Bio-Nano Science and
Technology, The University of New South Wales
Sydney, 2052, NSW (Australia)
E-mail: p.thordarson@unsw.edu.au
Prof. J. Wiedenmann
Coral Reef Laboratory/Institute for Life Science (IfLS)
University of Southampton, Southampton, SO14 3ZH/SO17 1BJ
(UK)
Prof. P. M. G. Curmi
School of Physics, The University of New South Wales
Sydney, 2052, NSW (Australia)
Dr. C. P. Marquis
School of Biotechnology and Biomolecular Sciences
The University of New South Wales, Sydney NSW 2052 (Australia)
C. K. Wong, Prof. M. H. Stenzel
School of Chemistry, Centre for Advanced Macromolecular Design
The University of New South Wales, Sydney, 2052, NSW (Australia)
[**] This work was supported by the Australian Research Council (ARC)
centre of excellence grant (grant number CE140100036) to P.T. and
J.J.G., an ARC Future Fellowship (FT120100101) to P.T., support
from the Faculty of Science UNSW to C.P.M. and P.T. and the
Australian Government for PhD scholarship to A.J.L.. C.K.W. is
grateful for an ARC and UNSW PhD scholarship.
To incorporate fluorescence in the polymersomes,
we selected
a green-fluorescent protein derived from
the reef coral species Acropora millepora (amilFP497).[22]
The 27.3 kDa protein is characterized by an absorption
and emission maxima of 477 and 497 nm, respectively. The
protein contains a free cysteine residue at position 119 for
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201412406.
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Communications
buffer (pH 7.4) containing 20 mm ethylenediaminetetracetic
acid (EDTA). Successful bioconjugation was validated by
matrix-assisted laser desorption/ionization time-of-flight
(MALDI-TOF) mass spectrometry and fast protein liquid
chromatography (FPLC).
The (MALDI-TOF) mass spectrometry (Figure 1A and
Figure S6) analysis shows a broad peak corresponding to the
bioconjugate at about 38500 gmolÀ1 (Figure 1A). The inten-
sity of the bioconjugate peak in the mass spectrum cannot be
used for quantitative analysis as the ionization of protein–
polymer bioconjugates has been known to be more difficult
than native proteins.[27] Interestingly, a small peak observed at
46000 gmolÀ1 (Figure 1A, inset) suggests that there may be
two PNIPAM chains attached onto the protein. This could
also explain why there is a small shoulder at the start of the
size-exclusion chromatograph in Figure 1B. Since the poly-
mer was used in excess to react with the protein, we propose
that some of the maleimide-bearing polymers may have
reacted with primary amines of surface lysine residues or even
additional cysteine residues buried inside the protein.
Further evidence of successful bioconjugation was
observed during the purification of the crude bioconjugate
Scheme 1. Polymersomes formed by self-assembly of PNIPAM-b-
amFP497 bioconjugates upon heating above the lower critical solution
temperature (LCST). The biohybrid polymersomes were found to be
capable of encapsulating a payload such as the red-fluorescent light-
harvesting protein (PE545) and a small organic drug molecule
(doxorubicin). However, encapsulation within the polymersomes
appeared to be size-dependent. The PE545 protein was found to be
encapsulated primarily within the membrane of the polymersomes
whereas doxorubicin was found to be encapsulated both within the
membrane and in the aqueous core of the polymersomes. The
polymersomes are depicted as unilamellar bilayer with the hydrophilic
(positively charged at pH 7) amilFP497 acting as the polar head-group
of the block copolymer. Note, however, that a multilayer membrane
structure cannot fully be ruled out.
site-selective bioconjugation with a maleimide-terminated
polymer.
In this study, we used poly(N-isopropylacrylamide)
(PNIPAM), a thermoresponsive polymer which undergoes
transitions from being hydrophilic to hydrophobic upon
heating above
a lower critical solution temperature
(LCST).[23] Olsen and co-workers have previously shown
that PNIPAM-fluorescent proteins are a powerful tool to
study protein–polymer block copolymer assembly.[24,25] We
envisaged that the use of this thermoresponsive polymer
would confer temperature responsiveness to both the bio-
conjugate and its resulting self-assembled structure.
The polymer with an estimated calculated molecular
weight (Mn,NMR) of 9420 gmolÀ1 and a degree of polymeri-
zation (DPn) of 80, was synthesized by reversible addition–
fragmentation chain transfer (RAFT) polymerization and
subsequently modified to bear a maleimide functional group
which readily reacts with a cysteine residue as shown in the
Supporting Information (Figures S1–S5).[26] The end-func-
tional maleimide group was identified by the presence of
a characteristic maleimide signal observed at d = 6.7 ppm in
Figure 1. A) MALDI-TOF mass spectrum of a desalted crude mixture of
maleimide-terminated PNIPAM and amilFP497 after reaction for 17 h.
The inset shows the enlarged region where the PNIPAM-b-amilFP497
peak was observed. B) Overlay of size-exclusion chromatography (SEC)
traces showing peaks corresponding to PNIPAM-b-amFP497 (dotted
line) and amilFP497 (solid line). The SEC column was eluted with
phosphate buffered saline (pH 7.4) and the detector was set to
monitor the absorbance at 477 nm.
1
the H NMR spectrum of the polymer (see Figure S1). The
dispersity index of the modified polymer was determined to
be 1.10 by gel permeation chromatography in N,N-dimethyl-
acetamide.
Conjugation of the maleimide-modified polymer to
amilFP497 was performed in 20 mm sodium phosphate
2
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reaction mixture by fast protein liquid chromatography
(FPLC). The FPLC size-exclusion chromatography (SEC)
trace of the crude bioconjugate mixture showed two peaks
corresponding to the elution of unreacted amFP497 and the
higher molecular weight PNIPAM-b-amilFP497, as shown in
Figure 1B. The conjugation efficiency was determined to be
40% by comparing the integrations of the two peak areas.
Minimal changes in the amilFP497 absorption and
emission spectra upon bioconjugation with PNIPAM (see
Figure S7) indicated that the amilFP497 proteins remains
folded and are still in their native state after bioconjugation.
To further investigate the thermoresponsive behavior of
PNIPAM-b-amilFP497, a cloud point profile of normalized
absorbance against temperature (see Figure S9) was plotted
and the lower critical-solution temperature (LCST) was
determined to be 36.5 Æ 0.58C—a transition ideally correlated
with the normal human body temperature.
The presence of the fluorescent protein in the bioconju-
gate allows the investigation of PNIPAM-b-amilFP497 self-
assembly using confocal microscopy. No distinct morphology
was observed in a solution of PNIPAM-b-amilFP497 biocon-
jugates analyzed at room temperature (see Figure S10).
However, upon heating to 378C, circular particles that
resemble polymersomes were observed, as shown in Fig-
ure 2A. The average external diameter and average mem-
brane thickness of the polymersomes were found to be 2085 Æ
297 and 290 Æ 36 nm, respectively (histograms in Figures S12
and S13). The measured thickness suggests these vesicles
might be multilamellar, however, given the resolution limit of
confocal microscopy is in the order of 250 nm, this value
cannot be relied upon. The hollow cores present within
individual polymersomes are revealed from a 3D representa-
tion obtained from a collection of 79 images across different
z heights (Figure 2B). Attempts to resize the polymersomes
by sonication were only moderately successful—the average
diameter increased to 2230 Æ 354 nm (Figures S14 and S15),
however the polydispersity also appeared to increase with
signs of protein degradation in the images.
Further evidence of the polymersome morphology was
obtained by transmission electron microscopy (TEM). The
TEM images showed spherical particles with an average
external diameter of 1292 Æ 184 nm (Figure S16), dark out-
lines, and distinct inner contrasts (Figure 2C), which is
consistent with polymersome formation.[28,29] The polymer-
somes observed by TEM appeared to have a smaller average
external diameter to those observed by CLSM but this
difference can easily be attributed to the drying effect during
the TEM specimen preparation. To further support our claims
regarding the size of these polymersomes, the average
hydrodynamic diameter (dh) of the polymersomes was studied
using dynamic light scattering (DLS) (Figure 2D). At 258C,
the PNIPAM-b-amilFP497 bioconjugates had a dh of 22 Æ
0.54 nm. When heated to 378C, particles with dh of 2005 Æ
211 nm and polydispersity index (PDI) of 0.21 were observed.
The dh of the polymersomes is in good agreement with the
polymersome sizes observed under CLSM.
To explore the potential of using PNIPAM-b-amilFP497
and related polymersomes as nanocarriers, encapsulation
experiments involving acceptor fluorophores were per-
formed. Successful encapsulation was determined by measur-
ing FRET from amilFP497 (located in the membrane
PNIPAM-b-amilFP497 polymersomes) to the encapsulated
fluorophores, using fluorescence lifetime imaging microscopy
(FLIM). We identified two suitable spectrally overlapping
fluorophores for the experiment; namely the light-harvesting
phycoerythrin 545 (PE545)[30,31] and a common anti-cancer
drug: doxorubicin hydrochloride (DOX). Both the excitation
spectra of PE545 and DOX overlap with emission spectra for
amilFP497 (see Figures S17 and S18 for spectral overlap).
Samples containing
a mixture of the PNIPAM-b-
amilFP497 and the FRET acceptors were initially imaged at
258C showing little to no energy transfer, and subsequently
heated to 408C to encapsulate an acceptor facilitating energy
transfer as the PNIPAM-b-amilFP497 donor came within
Fçrster distance of the acceptor, leading to a quenched
lifetime of the donor. Two detectors were used to investigate
the donor–acceptor system, one for the lifetime of the
PNIPAM-b-amilFP497 donor (485/32 nm—32 nm bandpass
filter centered at 485 nm) corresponding to the amilFP497
emission and another to measure the lifetime of the acceptors
(617/73 nm—73 nm bandpass filter centered at 617 nm)
focused on the acceptor emission spectra, with the lifetime
data plotted on a false color scale as shown in Figure 3. The
fluorescence lifetime data were analyzed using both expo-
nential tail fitting for the determination of lifetimes, and by
phasor analysis[32] to investigate the location of the lifetimes of
interest (see the Supporting Information for full analysis).
Initially, PNIPAM-b-amilFP497 was imaged as a control
(Figure S19, Table S1), and we found that the green fluores-
Figure 2. A) Confocal laser scanning microscopy (CLSM) image and
B) lateral view of a z-stack image of polymersomes formed from
PNIPAM-b-amilFP497 bioconjugates in 20 mm sodium phosphate and
20 mm EDTA buffer (pH 7.4) at 378C. The scale bar represents 2 mm.
C) Transmission electron microscopy (TEM) image (unstained) of
PNIPAM-b-amilFP497 incubated at 378C prior to analysis. D) Particle
size distribution from dynamic light scattering (DLS) of PNIPAM-b-
amilFP497 in 20 mm sodium phosphate, 20 mm EDTA buffer (pH 7.4)
at 25 and 378C. The scale bar represents 2 mm.
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Figure 3. Fluorescence lifetime imaging microscopy (FLIM) images of PNIPAM-b-amilFP497 polymersomes formed at 408C with encapsulated
doxorubicin (DOX, top row), phycoerythrin 545 (PE545, middle row), and a co-encapsulated mixture of DOX and PE545 (bottom row). Images in
columns A, B, C, and the overlay in E (B+C) were obtained using 405 nm excitation whereas images in column D were obtained using 532 nm
excitation. The relevant lifetime color scales (in nanoseconds) are shown under columns B–D while for the images in column E the colorscale is
shown to the left of these images. A) Gray-scale fluorescence image of 485/32 nm (32 nm bandpass filter centered at 485 nm) and 617/73 nm
(73 nm bandpass filter centered at 617 nm) channels overlaid (total emission). B) 485/32 nm channel corresponding to amilFP497 emission with
a lifetime colormap from 0 to 2.5 ns. C) 617/73 nm channel corresponding to the FRET-based acceptor emission with a colormap from 0.7
(similar to PE545) to 1.0 ns (similar to DOX). D) 617/73 nm channel corresponding to acceptor emission using 532 nm excitation with a colormap
from 0.7 (similar to PE545) to 0.9 ns (similar to DOX). E) Overlay of B+C; the amilFP497 emission in the 485/32 nm channel and the FRET
acceptor emission in the 617/73 nm channel (false red). Detailed lifetime analysis (Tables S2–S7) and phasor analysis of this data is in the
Supporting Information. The scale bar represents 2 mm.
cence of the fully formed PNIPAM-b-amilFP497 polymer-
somes had its lifetime reduced from 2.98 ns (258C) to 1.93 ns
(408C). This new lifetime is expected because of the close
proximity of neighboring amilFP497 proteins in the polymer-
some morphology which leads to self-quenching of
amilFP497. Importantly, in the presence of the acceptors,
the formation of polymersomes was not hindered, yet some
minor differences in polymersome morphology were
observed (Figure 3 and Figures S19–S21).
the acceptors, the samples were additionally excited using
a 532 nm laser allowing no excitation of the donor, which
again showed the membrane localization of the proteins, and
the aqueous core and membrane locations of DOX (Fig-
ure 3D). Proteins are known to adsorb/adhere onto PNIPAM
at temperatures well above the LCST[33,34] and it is therefore
not surprising to find the light-harvesting PE545 protein in the
polymersome membrane.
Further, once the acceptors were encapsulated,
Table 1: Fluorescence lifetimes based on exponential fitting of data from FLIM
images (Figure 3 and Tables S2, S4, and S6) of PNIPAM-b-amilFP497 polymersomes
encapsulating doxorubicin (DOX), the phycoerythrin 545 (PE545) protein and, the
co-encapsulation of DOX+PE545.
the lifetime of the PNIPAM-b-amilFP497 polymer-
somes was reduced from 1.93 to 1.32 ns for DOX and
0.29 ns for PE545 (Table 1, Figures S20 and S21,
Tables S2–S5).
[a]
[b]
[c]
[d]
Sample
t1
t2
a1
a2
c2[e]
E[f]
We found that the PNIPAM-b-amilFP497 poly-
mersomes were capable of selectively encapsulating
molecules within different regions of the polymer-
some structure depending on their properties. The
light-harvesting PE545 protein acceptor was primar-
ily located in the membrane of the polymersomes
whereas the small drug molecule (DOX) was pri-
marily encapsulated in the aqueous core but also
PNIPAM-b-amilFP497+DOX
PNIPAM-b-amilFP497+PE545
PNIPAM-b-amilFP497+PE545+DOX 0.30 1.86 97
1.32 0.66 53
0.29 1.86 83
47
17
3
1.46 32%
2.66 85%
2.60 85%
[a] Primary fluorescence lifetime. [b] Secondary fluorescence lifetime. [c] Primary
lifetime pre-exponential percentage (characterizes the population of the primary
lifetime species). [d] Secondary lifetime pre-exponential percentage (characterizes
the population of the secondary lifetime species). [e] Fitting error in the exponential
decay. [f] FRET efficiency calculated using E=1À[t(donor in the presence of
found in the membrane. To confirm the location of acceptor)/t(donor in the absence of acceptor)].
4
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Interestingly, we can also co-encapsulate DOX and the
PE545 protein in the same polymersome but resolve their
spatial location by FLIM-FRET (Figure 3—bottom row) as
the DOX and PE545 lifetimes differ quite significantly. The
fluorescence lifetime maps in both the FRET (Figure 3C) and
acceptor excitation—acceptor emission (Figure 3D) chan-
nels, show that the PE545 protein is localized in the
membrane of the polymersomes with DOX in the interior
(and presumably membrane, although the excess PE545
masks most of the DOX emission)—in line with when these
molecules were loaded on their own into these polymersomes.
The phasor analysis (Figure S22) and exponential tail fitting
(Table S7) further confirmed this, showing the two acceptor
lifetimes; about 0.4 ns from PE545 and about 1.4–1.5 from
DOX. These results illustrate the power of the FLIM-FRET
approach in monitoring the fate of payload mixture in
a polymersome.
There is not a large difference in the diameter of our
empty PNIPAM-b-amilFP497 polymersomes compared to
those loaded with DOX, PE545 or mixtures of both (compare
Figure S12 vs. Figures S23–S25) and the cloud point only
increases by about 3–58C (Figure S9 vs. Figure S26). The
PE545 and DOX protein-loaded polymersomes are about 10–
20% smaller in diameter than the empty polymersomes,
suggesting that the cargo improves the packing of the
PNIPAM-b-amilFP497 in these polymersomes.
Keywords: compartmentalization · encapsulation · FRET ·
polymersomes · protein modifications
.
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We have shown here that the conjugation of PNIPAM
with amilFP497 results in the thermoresponsive PNIPAM-b-
amilFP497 block copolymer that forms well-defined poly-
mersomes upon heating to 378C. If these polymersomes are
formed in the presence of suitable fluorescent acceptors for
the amilFP497 protein, the encapsulation of these acceptors
can be spatially resolved using FLIM-FRET. Using this
microscopy-based approach it was shown that while the
water-soluble drug DOX was encapsulated in both the core
and the membrane of the PNIPAM-b-amFP497 polymer-
somes, the light-harvesting PE545 protein was exclusively
bound in the polymersome membranes. Importantly, we were
also able to show that the PE545 protein and DOX could be
co-encapsulated in these polymersomes with the resulting
lifetime maps clearly revealing their different location in the
polymersome membrane and core, respectively. These results
demonstrate the capability of FRET-FLIM to study the
encapsulation of (bio)molecular payloads by carefully design-
ing the fluorescent properties of the polymersomes. Block
copolymers based on fluorescent protein–polymer conjugates
are particularly attractive in this regard as encapsulated
proteins and protein–polymer bioconjugate are frequently
used in functional polymersomes. We envision that this
approach will be of generic use in polymersome research
including in the monitoring of drug release and in the study of
complex compartmentalized self-assembled structures such as
vesicles-in-polymersomes.
Received: December 26, 2014
Revised: February 5, 2015
Published online: && &&, &&&&
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Angewandte
Communications
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6
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Angew. Chem. Int. Ed. 2015, 54, 1 – 7
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Angewandte
Chemie
Communications
Polymersomes
C. K. Wong, A. J. Laos, A. H. Soeriyadi,
J. Wiedenmann, P. M. G. Curmi,
J. J. Gooding, C. P. Marquis,
M. H. Stenzel,
P. Thordarson*
&&&& — &&&&
Polymersomes Prepared from
Temperature-induced self-assembly: A
temperature-sensitive green fluorescent
(amilFP497) protein–polymer bioconju-
gate forms polymersomes above 378C
which encapsulate a mixture of pink
fluorescent protein (PE545) and a red
Thermoresponsive Fluorescent Protein–
Polymer Bioconjugates: Capture of and
Report on Drug and Protein Payloads
drug molecule (DOX). The spatial loca-
tion of the payload is revealed by fluo-
rescence lifetime microscopy.
Angew. Chem. Int. Ed. 2015, 54, 1 – 7
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
7
These are not the final page numbers!