Letter
pubs.acs.org/NanoLett
Aiding Nature’s Organelles: Artificial Peroxisomes Play Their Role
Pascal Tanner, Vimalkumar Balasubramanian, and Cornelia G. Palivan*
Department of Chemistry, University of Basel, Klingelbergstrasse 80, CH-4056 Basel, Switzerland
S Supporting Information
*
ABSTRACT: A major goal in medical research is to develop
artificial organelles that can implant in cells to treat pathological
conditions or to support the design of artificial cells. Several
attempts have been made to encapsulate or entrap enzymes,
proteins, or mimics in polymer compartments, but only few of
these nanoreactors were active in cells, and none was proven to
mimic a specific natural organelle. Here, we show the necessary
steps for the development of an artificial organelle mimicking a
natural organelle, the peroxisome. The system, based on two
enzymes that work in tandem in polymer vesicles, with a
membrane rendered permeable by inserted channel proteins was
optimized in terms of natural peroxisome properties and
function. The uptake, absence of toxicity, and in situ activity in cells exposed to oxidative stress demonstrated that the
artificial peroxisomes detoxify superoxide radicals and H2O2 after endosomal escape. Our artificial peroxisome combats oxidative
stress in cells, a factor in various pathologies (e.g., arthritis, Parkinson’s, cancer, AIDS), and offers a versatile strategy to develop
other “cell implants” for cell dysfunction.
KEYWORDS: Polymer nanoreactor, artificial organelle, antioxidant enzymes, reactive oxygen species, peroxisome
C
synthetic membranes10 or by the insertion of channel
proteins12 depending on the chemical nature of the copolymer.
Even though a variety of nanoreactors that support specific
reactions has been introduced,6,11−13 only recently have these
been tested for uptake in cells. In a very few studies,
nanoreactor activity inside cells pointed to behavior such as
could be expected from a simple, artificial organelle.14−17
However, none has been proposed to mimic one of the existing
cell organelles.
To design mimics of cell organelles, complex requirements
need to be fulfilled:18 (i) polymer compartments of appropriate
size for cell-uptake, (ii) activity of encapsulated enzyme(s)
maintained in situ, (iii) a polymer membrane, which is stable
and permeable to small molecules, (iv) integrity and activity
preserved in cells (i.e., an artificial organelle), and (v)
biocompatibility and biodegradability of polymer compartments. This complexity could explain why only very few
examples of nanoreactors have been reported to be integrated
and active in cells.
We recently introduced the concept of an antioxidant
nanoreactor in which enzymes/enzyme mimics encapsulated
in polymer vesicles successfully detoxified superoxide radicals14,19 and peroxynitrites,20 well-known reactive oxygen
species (ROS) involved in oxidative stress. Beyond that, in
THP-1 cells exposed to oxidative stress, two types of enzymes
reating artificial organelles that aid their natural counterparts in cells will have a dramatic impact on medicine in
the treatment of disorders and in the design of artificial cells.
Because organelles are key components in cells and comprise
compartments loaded with molecules essential to life, their
inadequate functioning can contribute to numerous pathological conditions.1 Scientists have already anticipated that the
design of molecular factories, such as artificial organelles, will
provide a new frontier in medicine.2 To this end, nanoscience
provides a necessary tool, due to its specificity in the
development of structures and assemblies that are compatible
with the size range of natural organelles. In particular,
amphiphilic copolymers, which self-assemble into supramolecular structures3 such as micelles, tubes, and vesicles,
represent ideal candidates to form organelle-like compartments
that can contain combinations of biomolecules, because their
chemistry allows the adjustment of properties such as stability,
flexibility, and functionality.4 For example, polymer vesicles5
have been reported as nanocarriers for active compounds6
ranging from small molecular weight drugs to proteins or DNA,
with improved stability as compared to lipid vesicles.2 They can
be functionalized at the outer surface for targeting approaches7
and possess stimuli-responsiveness8 for release of encapsulated
compounds “on demand”. A step beyond this was realized by
the design of nanoreactorspolymer compartments that
encapsulate active compounds (enzymes, proteins, enzyme
mimics) with the dual role of protecting the compounds from
proteolytic attack and serving as a reaction space.9 The
necessary exchange of substrates/products with the environment to support in situ reactions occurs through porous,
© 2013 American Chemical Society
Received: April 4, 2013
Revised: May 2, 2013
Published: May 6, 2013
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cascade reaction and increasing their encapsulation efficiency
serves to control the overall activity of an AP. We evaluated the
processes by which an AP is taken-up and distributed, such as
internalization kinetics, intracellular trafficking, and translocation, to determine functional aspects of an artificial
peroxisome. The design of artificial organelles, such as our
AP, will aid in the production of “cell implants” that can
contribute to the treatment of various cell disorders and will
ultimately support the design of artificial cells. In addition, the
strategy we introduce here for the development of APs from
the molecular level onward would provide the opportunity to
tailor different combinations of active compounds (biomolecules, mimics, and drugs), thereby providing solutions for
personalized medicine.
Results and Discussion. Design of Artificial Peroxisomes.
To produce an artificial peroxisome (AP) that shares the
functioning and properties of a natural peroxisome, we
developed and optimized the gated polymer vesicle in which
a cascade reaction can be used to combat reactive oxygen
species (ROS)16 (Figure 1D). To enhance the overall cascade
reaction occurring as tandem activity of Cu/Zn-superoxide
dismutase (SOD), the first enzyme, and lactoperoxidase
(LPO), the second enzyme, we increased the enzymes
encapsulation efficiency and changed LPO with catalase
(CAT). In nature, SOD detoxifies superoxide radicals28 to
H2O2 via a two-step reaction, while the second enzyme, LPO29
or CAT,30 serves to degrade H2O2 to harmless products,
molecular oxygen and water. We selected the poly(2methyloxazoline)-b-poly(dimethylsiloxane)-b-poly(2-methyloxazoline), (PMOXA-PDMS-PMOXA) triblock copolymer to
produce AP compartments. It has been shown that this type of
amphiphilic polymer self-assembles in dilute solution and
generates vesicles of high mechanical stability,31 sizes in the
nanometer range, and features none of the organizational
defects responsible for the known instability of liposomes.
Vesicle membranes of PMOXA-PDMS-PMOXA copolymer are
permeable to superoxide radicals and oxygen,19 while they are
impermeable to other molecules of higher molecular weight,
such as glucose, glycerol, and urea.32 We chose to incorporate
the outer membrane protein F (OmpF), which allows passive
transport of molecules up to 600 Da,33 to serve as a gate for the
substrates/products of our cascade reaction, while the enzymes
cannot escape from the vesicle cavity. Channel protein insertion
in synthetic membranes is an elegant approach to the
development of mimics of a biomembrane.
We used a direct dissolution method for AP preparation
based on the chemical nature of PMOXA-PDMS-PMOXA
copolymers and to avoid organic solvents (Supporting
Information). Such mild encapsulation conditions have already
been reported to preserve the integrity and activity of a variety
of enzymes encapsulated in nanoreactors.16,19,20 Variation of
the hydrophilic-to-hydrophobic ratio of PMOXA-PDMSPMOXA copolymers served to optimize the supramolecular
structures generated by self-assembly in terms of architecture
(vesicles as major population of assemblies) and size.
Homogeneous, empty PMOXA12-PDMS55-PMOXA12 polymer vesicles at a diameter of 200 ± 40 nm, which is comparable
to the size range of a peroxisome from 200 nm to 1.1 μm,34
were generated in buffer by the self-assembly process, as
established by dynamic and static light scattering (Supporting
Information, Figure S1). The formation of vesicles was
confirmed by transmission electron microscpy (TEM; Figure
2A). The larger value of the diameter of vesicles based on LS
that acted in tandem within polymer vesicles degraded
superoxide radicals and related H2O2.16
Here, we go one step further, providing evidence that an
enzyme-containing polymer vesicle mimics a natural cell
organelle (Figure 1A), the peroxisome (Figure 1B), upon
Figure 1. (A) Schematic view of a cell showing various organelles. (B)
Peroxisomes are spherical, cell organelles of sizes in the nanometer
range that play a significant role in the regulation of ROS. (C) An
artificial peroxisome (AP) is based on the simultaneous encapsulation
of a set of antioxidant enzymes in a polymer vesicle, with a membrane
equipped with channel proteins. The APs serve for ROS detoxification
inside cells. (D) Schematic enzymatic cascade reaction occurring
inside the AP and serving to detoxify superoxide radicals and related
H2O2.
optimization in terms of properties and function. A peroxisome
is a multipurpose, cell organelle involved in fatty acid αoxidation, the catabolism of purines, the biosynthesis of
glycerolipids, and the regulation of reactive oxygen species
(ROS) at the molecular level.21 Peroxisomes are nanometerscale, round, vesicle-like organelles that contain various
antioxidant enzymes such as catalase, glutathione peroxidase,
and Cu/Zn- or Mn-superoxide dismutase. Cellular antioxidant
defense mechanisms, to which peroxisomes contribute, can be
overwhelmed by an imbalance in ROS associated with oxidative
stress. Increased levels of ROS, which contribute to the
pathogenesis of various diseases (e.g., arthritis, Parkinson’s
disease, cancer, and AIDS),22−25 and are responsible for
inorganic nanoparticle toxicity,26 represent a serious concern
in medicine. Thus, the design of artificial peroxisomes is
expected to represent an excellent solution in this regard.
Our artificial peroxisome (AP) is based on the tuned coencapsulation of two antioxidant enzyme types that support a
specific cascade reaction in the cavity of a polymer vesicle. The
vesicle membrane is provided with channel proteins that serve
as gates for substrates and products (Figure 1C and D). The
advantage of using the bottom-up approach of combining
polymer compartments with active molecules serve for the
optimization of an AP with respect to: (i) compartment
properties, (ii) enzyme co-encapsulation efficiency, and (iii)
enhancement of the cascade reaction. Optimization of
compartment properties is necessary to improve cell integration
of an AP.27 Substituting the types of enzymes involved in a
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Figure 2. (A) TEM micrograph of empty PMOXA-PDMS-PMOXA vesicles. (B) TEM micrograph of LPO-encapsulated vesicles. (C) TEM
micrograph of SOD-LPO-containing APs. Scale bars = 200 nm. (D) APs solution used for cell-uptake studies. (E) FCS autocorrelation curves and
fits of: (a) AlexaFluor-633, (b) LPO-AlexaFluor-633, and (c) LPO-AlexaFluor-633-containing vesicles. (F) FCS and FCCS of APs: (a)
autocorrelation curve of vesicles containing SOD-AlexaFluor-488 together with its fit; (b) autocorrelation curve of vesicles containing LPOAlexaFluor-633 together with its fit, and (c) cross-correlation curve of SOD-AlexaFluor-488 and LPO-AlexaFluor-633 co-encapsulated in APs
together with its fit. Dotted lines are experimental curves.
experiments compared to TEM is expected, as the DH from
DLS experiments represents the sum of the vesicle and its
surrounding hydration sphere. To obtain a membrane with
high permeability while keeping the vesicles intact, we gradually
increased the OmpF concentration initially used for insertion.35
A final concentration of OmpF of 0.05 μg/μL proved optimal
for membrane permeability and did not affect membrane
stability.
The design of an AP first requires characterization and
optimization of the encapsulation of each enzyme type in a
polymer vesicle to provide the best conditions prior to enzyme
co-encapsulation. An increase in the initial concentration of
each enzyme to achieve higher encapsulation efficiency had to
be balanced with respect to vesicle aggregation or disturbing
the self-assembly process of vesicle formation. The initial
enzyme concentration used for enzyme encapsulation did not
affect the morphology or size of the nanoreactors, as established
by a combination of light scattering and TEM for: SOD
(Supporting Information, Figure S2), LPO (Figure 2B,
Supporting Information, Figure S3), and CAT (Supporting
Information, Figure S4).
Each enzyme was labeled with a different fluorescent dye
(AlexaFluor-488 for SOD, AlexaFluor-633 or dylight-633 for
LPO, and dylight-488 for CAT), and its encapsulation
efficiency was established by a combination of fluorescence
correlation spectroscopy and brightness measurements (Supporting Information). A labeling efficiency of 3 AlexaFluor-488
molecules per SOD, 1 AlexaFluor-633 molecule, or 2.7 dylight633 molecules per LPO, and 1 dylight-488 molecule per CAT
was obtained. The number of enzymes per vesicle was
determined by dividing the value of the molecular brightness
of enzyme-containing vesicles, expressed as counts per
molecule (CPM), by the CPM of freely diffusing, labeled
enzymes. We calculated the encapsulation efficiency in vesicles
(EEv) by dividing the number of enzyme molecules per vesicle
by the maximum number of enzyme molecules possible to be
encapsulated inside a vesicle via a statistical, self-assembly
process of encapsulation, based on the initial enzyme
concentration (Supporting Information, eqs S1−S4).36
The change in the diffusion time, from the value characteristic for free SOD-AlexaFluor-488 (τD = 109 μs) to a value of
3.5 ± 1 ms, indicates that the enzyme was encapsulated in
vesicles (Supporting Information, Figure S5).19 Based on
various initial amounts of SOD-AlexaFluor-488 (0.1−0.5 mg/
mL), an optimum EEv of 25% ± 10% was obtained using an
initial enzyme concentration of 0.25 mg/mL (Supporting
Information, Figure S5). To exclude unspecific binding by
SOD-AlexaFluor-488 or AlexaFluor-488, which would lead to
greater uncertainty in the number of encapsulated enzymes,
empty polymer vesicles were incubated with dye-labeled
enzyme or a dye molecule. In both cases, no unspecific binding
was detected. A further increase in the initial SOD
concentration used for encapsulation of up to 0.9 mg/mL
was not possible, due to the intrinsically poor solubility of SOD.
However, because SOD has very high reaction kinetics (kcat ≈
10−9 M−1 s−1),37 only a few enzymes per vesicle were sufficient
to support the in situ cascade reaction.16
Similar to SOD, the successful encapsulation of LPOAlexaFluor-633 in polymer vesicles was indicated by a change
in the diffusion time from the value corresponding to the free,
labeled-LPO (τD = 320 μs) to a value of 8.5 ± 1 ms, which
corresponds to the enzyme-containing vesicles. Various initial
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Figure 3. (A) Viability in HeLa cells after incubation with different concentrations of APs (MTS assay). Blue bars: incubation time 24 h, red bars:
incubation time 48 h. (B) Confocal laser scanning microscopy (CLSM) images of HeLa cells incubated with APs containing SOD-AlexaFluor-488
and LPO-AlexaFluor-633 for 24 h (scale bar = 20 μm). (C) CLSM images of HeLa cells incubated with APs containing SOD and LPO-AlexaFluor633 for different incubation times (scale bar = 20 μm). (D) Flow cytometry analysis of APs in HeLa cells for different incubation times (0, 2, 4, 8, 18,
24 h).
cases where no aggregates were present and the change of the
dye did not improve the EEv of LPO, we used only LPOAlexaFluor-633 for AP characterization.
The bottom-up approach to build an AP allows an exchange
of enzyme types to optimize the cascade reaction in situ. We
tested CAT instead of LPO as the second enzyme, to
determine whether we would obtain an increased overall
activity of the APs. The advantage of CAT is that it allows
simplified conditions for the assessment of the overall activity
of APs, because if detoxifies H2O2 in a catalytic reaction,
without the necessity of additional cosubstrates.30 However, as
CAT has a higher molecular weight (250 kDa),30 it creates
problems for encapsulation efficiency, as shown before with
lipid vesicles.38 The change in the diffusion time from the value
corresponding to the free CAT-dylight-488 (τD = 340 μs) to a
value of around 4 ± 1 ms, indicates that CAT-dylight-488 was
successfully encapsulated in polymer vesicles. No aggregates
were present across the entire range of initial enzyme
concentrations (Supporting Information, Figure S9). An EEv
of 16% ± 9%, significantly lower compared to that of LPO, was
amounts of LPO-AlexaFluor-633 (from 0.1 mg/mL to 0.5 mg/
mL) have been tested to optimize enzyme encapsulation
efficiency. An optimum EEv of 66% ± 33% was obtained using
an initial LPO-AlexaFluor-633 concentration of 0.25 mg/mL
(Figure 2E, Supporting Information, Figure S6). The high EEv
of LPO leads to the final activity of the APs. At higher initial
concentrations of LPO-AlexaFluor-633 used for the encapsulation, for example 0.83 mg/mL, aggregates with a diffusion
time of >30 ms were determined by FCS, and confirmed by
TEM (Supporting Information, Figure S7). A diffusion time of
around 7 ms was obtained when empty vesicles were measured
in the presence of the free dye (50 nM). This unspecific
binding of AlexaFluor-633 to the PMOXA 12 -PDMS 55 PMOXA12 vesicles explains the presence of large aggregates
formed at the high, initial LPO-AlexaFluor-633 concentrations
used for encapsulation. As expected, the labeling to LPO with a
different dye, dylight-633, prevented the formation of
aggregates when high initial concentrations of LPO were
used for the encapsulation procedure (Supporting Information,
Figure S8). However, because the optimum EEv of LPO was
obtained at a significantly lower initial enzyme concentration, in
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Figure 4. (A) CLSM image of HeLa cells incubated for 24 h with APs (blue: nucleus stained with Hoechst, green: endosome/lysosome stained with
pH-rodo, red: artificial peroxisomes) (scale bar = 5 μm). (B) TEM images of the peri-nuclear region of HeLa cells incubated with APs for 12 h (scale
bar = 200 nm). (C) TEM images of endosome-like compartments in HeLa cells incubated with APs for 12 h (Scale bar = 200 nm). (D) Protective
effect of APs against paraquat by flow cytometry analysis: viability of untreated cells considered as 100%, (cells), viability of cells after treatment with
paraquat (PQ) and viability of cells pretreated with APs for 24 h, and sequentially treated with paraquat for a further 24 h (PQ-APs). (E) Real time
ROS detoxification kinetics of APs in: (a) cells treated with pyocyanin, and (b) cells pretreated with APs (8 h) followed by treatment with
pyocyanin.
calculated for CAT encapsulation based on brightness
measurements correlated with FCS.
We compared the enzymatic kinetics of LPO-containing
vesicles to that of CAT-containing vesicles (Supporting
Information, Scheme S1). For similar concentrations of LPO
and CAT under free conditions and adjusted to the
concentration range expected for APs, CAT competed with
LPO with comparable kinetics (Supporting Information, Figure
S10A). The equivalence of kinetics between peroxidase and
catalase under physiological conditions at low substrate
concentrations has already been reported.39 However, because
of the lower encapsulation efficiency of CAT compared to
LPO, H2O2 degradation by CAT-containing vesicles was less
effective (Supporting Information, Figure S10B). This indicates
that, under the given conditions of the APs, the key
determinant of overall activity is encapsulation efficiency, and
not the specificity of the enzyme. We chose to continue the
development of APs by including the combination of SOD and
LPO.
Fast, efficient ROS detoxification by APs requires successful
co-encapsulation of SOD and LPO in polymer vesicles with
OmpF-inserted membranes. The co-encapsulation process,
leading to a turbid solution of APs (Figure 2D), influenced
neither the morphology nor the size of vesicles, according to
TEM (Figure 2C) and light scattering (Supporting Information, Figure S11). Fluorescence cross-correlation spectroscopy
(FCCS) was used to establish the degree of simultaneous coencapsulation of fluorescently labeled SOD and LPO via the
statistical process of self-assembly (Figure 2F). The positive
cross-correlation curve (Figure 2F-c) was compared with the
autocorrelation curves for each enzyme (Figure 2F-a,b) and
proved the successful co-encapsulation of SOD-AlexaFluor-488
and LPO-AlexaFluor-633 in vesicles. Fluorescence brightness
measurements performed in each detection channel showed
that the ratio of SOD:LPO of 1:2 inside vesicles permitted the
cascade reaction (Figure 1D), as in the case of single-enzyme
encapsulation conditions. Cross-talk in the FCCS experiment
was negligible (<0.5 kHz), because of the well-separated
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available for uptake, and a possible saturation of cell storage
capacity (Supporting Information, Figure S13).42,43 In contrast,
APs generated at a high initial concentration of LPOAlexaFluor-633 showed a dramatic change in uptake behavior:
at a short incubation time of 2 h, approximately 60% of APs
were taken up due to aggregation, as expected (Supporting
Information, Figure S14).
The influence of aggregation on the uptake of nanoparticles
generally depends on the mechanism of the internalization
pathway.44 We analyzed the uptake mechanism of APs using
specific endocytosis pathway inhibitors: at least two different
pathways were involved, clathrin-mediated endocytosis and
macropinocytosis. As macropinocytosis is the dominant uptake
pathway involved in nonspecific uptake, this explains why AP
aggregation strongly affected cell uptake kinetics (Supporting
Information, Figure S15).45,46
Endosomal escape is a critical requirement if nanoreactors
are to serve as APs inside cells. In order to analyze whether APs
escape endosomes or are trafficked for degradation, we used an
endolysosome marker, pH-rodo, and visualized cells by CLSM.
The majority of APs were found in intracellular regions, in
particular the peri-nuclear region, and only very few were found
in endolysosome compartments (Figure 4A). The fluorescent
signals from the peri-nuclear region indicate that APs escaped
from endosomes. Co-localization signals of endolysosome
compartments and APs suggest that APs were transported
through endocytosis. To gain further insight into endosomal
escape, the intracellular localization of APs was visualized by
TEM. The TEM micrograph shows after 12 h the presence of
intact APs in peri-nuclear regions, with a few collapsed APs
(Figure 4B). APs were far fewer in number in endosome-like
compartments (Figure 4C). Intact APs escaped from endosomes and were distributed in the cytoplasm, in agreement with
the CLSM co-localization results. Escape from endosomes by
PMOXA-PDMS-PMOXA vesicles has already been reported in
THP-1 cells.14 To understand interfacial effects of triblock
copolymers on cell membranes, polymer−lipid interactions
were studied with PMOXA-PDMS-PMOXA copolymers. Small
amounts of PMOXA-PDMS-PMOXA copolymers were
sufficient to interact with lipid bilayers and determine a phase
separation followed by domain formation.47 Therefore, we
hypothesize that APs escape from endosomes due to the ability
of this type of polymer vesicle to induce phase separation of the
lipids in the endosomal membrane. However, we observed no
APs escape from the cells even after 48 h.
The functionality of APs in cells was assessed by two
strategies: (i) estimation of the overall protective effect of APs
on cells exposed to paraquat-mediated oxidative stress, and (ii)
real time monitoring of AP activity in live cells exposed to
pyocyanin-mediated ROS stimulation. In the first approach, cell
injury was produced by treatment of HeLa cells with paraquat,
known to induce severe effects up to apoptosis at high doses.48
The viability of the cells was quantified by PI staining and
analyzed with flow cytometry. Cells pretreated with APs
provided an overall protective effect of around 26% against the
paraquat-mediated oxidative stress in HeLa cells (Figure 4D).
Increased viability represents proof of the efficient, in situ
cascade reaction carried out by APs.
To determine the involvement of APs in ROS detoxification
directly in cells, it is important to monitor ROS generation in
intact, live cells. Such detection is difficult, due to the low
concentration and high reactivity of ROS. The short half-life of
ROS requires immediate, real time monitoring of live cells.49 In
emission spectra of AlexaFluor-488 and AlexaFluor-633.
Approximately 22% ± 10% of vesicles contained both enzymes
simultaneously, in agreement with the theoretical fraction of
enzyme-co-encapsulating vesicles that are produced by a selfassembly process.
Integration of APs in Cells. Designing efficient artificial
peroxisomes requires an understanding of their integration into
cells and the influence of their properties on cell interactions.
To support cell integration and the functioning of an AP, a
complex scenario of molecular events has been studied in detail
in cells by assessing: (i) toxicity, (ii) cell uptake of APs, (iii)
intracellular trafficking and translocation, and (iv) AP
functionality under intracellular conditions. First, the toxicity
of APs was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
(MTS) assay in the HeLa cell line. APs exhibit minimal in vitro
cytotoxicity in HeLa cells for a period of up to 48 h (Figure
3A). This agrees with the reported nontoxicity of PMOXAPDMS-PMOXA-based copolymers with different block lengths,
as studied in other cell lines.14,40
For integration into cells, the first level of cell barriers
encountered by an AP is interaction with the cell membrane,
followed by uptake. We evaluated uptake into HeLa cells of
APs containing co-encapsulated SOD-AlexaFluor-488 and
LPO-AlexaFluor-633. Co-localization of a green fluorescence
signal corresponding to SOD and a red fluorescence signal
corresponding to LPO indicated that APs containing both
enzymes were internalized and crossed the first cell barrier
(Figure 3B). Although two different enzyme types had been
loaded into polymer vesicles earlier by us and others,10,16 this is
the first proof of the presence of vesicles containing enzymes
that act in tandem in live cells. Optimization of APs in terms of
EEv correlated with the result observed inside cells: the amount
of SOD inside APs was only slightly increased compared to that
of LPO (Supporting Information, Figure S12). This resulted in
a significantly lower intensity of the fluorescence signal
corresponding to SOD as compared to that of LPO. Due to
this difference in fluorescence intensity, for the sake of
simplicity we performed uptake kinetics analyses in cells with
APs containing nonlabeled SOD and labeled LPO. As expected,
cell uptake increased with time, from 2 to 18 h, indicating timedependent internalization of APs (Figure 3C). Note that
fluorescence intensity varied from cell to cell, suggesting the
number of internalized APs in individual cells was different, due
to nonspecific interactions and the random number of APs
contacting cells at close proximity.
In general, cell uptake is directly related to the concentration
of nano-objects (nanoparticles, vesicles, etc). In addition, it has
been reported that the aggregation of nanoparticles dramatically influences cell uptake by a local increase in the
concentration to which cells are exposed as compared to the
initial, bulk concentration.41 To analyze the effect of AP
aggregation, we compared the uptake kinetics of APs generated
with a high initial concentration of LPO (0.83 mg/mL), known
to form aggregates (Supporting Information, Figure S7), to that
of APs generated with the optimum, initial concentrations of
enzymes (0.25 mg/mL). The mean cell fluorescence, which
corresponds to the overall cell population in a flow cytometer,
indicates that around 10% of APs containing LPO-AlexaFluor633 (0.25 mg/mL) were taken up after 2 h. The fraction of
internalized APs increased linearly over time up to 80% after 18
h (Figure 3D, Supporting Information, Figure S13). A plateau
was reached at longer periods of time, due to a depletion of APs
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Cytotoxicity. Cells were cultured in a 96-well plate at a
density of 2 × 104 cells/well and incubated with 100 μL of
DMEM growth medium per well for 24 h. Different
concentrations of empty polymer vesicles and enzyme-loaded
polymer vesicles were added to different wells and incubated
with cells for a period of time (24 and 48 h). Subsequently, 20
μL of CellTiter 96 Aqueous One Solution Cell Proliferation
Assay (MTS) reagent (Promega) were added to each well and
then incubated for a further 2 h. The absorbance of each well
mixture was measured with a Spectromax M5e microplate
reader (Molecular Devices) at 490 nm.
Cell Uptake. HeLa cells were cultured in a 6-well plate at
different densities of cells per well (1 × 105 for CLSM, 2 × 105
for flow cytometry analysis, and 1 × 106 for TEM) in DMEM
medium and incubated with enzyme-loaded polymer vesicle
solution at different time intervals (2, 4, 8, 18, 24 h) at 37 °C in
a humidified CO2 incubator. For flow cytometry analysis, cells
incubated with a solution of enzyme-loaded polymer vesicles
were washed with phosphate-buffered saline (PBS) to remove
free enzyme-containing vesicles and quantitatively analyzed for
rate of uptake of enzyme-containing vesicles with a flow
cytometry setup (CYAN, BD Biosciences). A total of 10 000
events was analyzed for each condition.
APs Escape from Endosome. HeLa cells preincubated with a
polymer vesicle solution containing co-encapsulated SOD and
LPO were fixed with a solution of 3% paraformaldehyde in PBS
and 0.5% glutaraldehyde in PBS containing 1.5% saccharose
(pH 7.3) and then postfixed in 0.5% aqueous osmium tetroxide.
The samples were then dehydrated using a graded series of
ethanol solutions, block-stained in 6% of uranyl acetate and
embedded in LR white resin (Sigma Aldrich). Ultrathin
sections were contrasted with 0.3% lead citrate and imaged
with TEM.
For CLSM visualization, the HeLa cell culture was first
treated with enzyme-loaded polymer vesicles (100 μg/mL) for
4 h, followed by treatment with pH-rodo Red dextran (20 μg/
mL) for a further 20 min at 37 °C following the supplier’s
protocol. The cells were rapidly washed with PBS and prepared
for visualization.
Cellular ROS Detoxification by APs. HeLa cells were treated
with paraquat to induce oxidative stress and analyzed by flow
cytometry (CYAN, BD Biosciences). HeLa cells at a density of
1 × 105 plated in a 6-well plate were first incubated with
enzyme-loaded polymer vesicles (500 μg/mL) for 24 h and
subsequently with paraquat (2 mM) for a further 24 h at 37 °C
in a 5% CO2 atmosphere. Cells were washed with PBS buffer
and stained with propidium iodide (PI, 50 mg/mL) to
determine cell viability. A total of 10 000 events were analyzed
for each condition. To analyze the ROS detoxification kinetics
of artificial peroxisomes in live cells, HeLa cells were seeded at
4 × 103 cells/well in a 96-well plate incubated overnight for cell
attachment, followed by incubation with APs vesicles for 8 h.
Afterward, cells were treated with ROS/superoxide detection
mix from Enzolife’s ROS/superoxide detection kit for 1 h. Cells
were then incubated with pyocyanin (300 μM) to induce ROS
generation in the cells. ROS detoxification kinetics was
measured with a Spectromax M5e microplate reader (Molecular Devices) in the kinetic mode with a 550/620 nm filter set.
the second approach, live cell analysis (4000 cells) of the
kinetics of ROS detoxification by APs was monitored in real
time with highly sensitive ROS fluorescent probes (Enzo Life
Sciences). Pyocyanin served to induce oxidative stress mediated
by ROS generation and to suppress the host antioxidant
mechanisms.50 An optimized concentration of pyocyanin
stimulated the continuous generation of ROS in live HeLa
cells. ROS were detoxified only in the case of stimulated cells
pretreated with APs (Figure 4E). The multipoint scans within
each well indicated an inhomogeneous intensity distribution of
the fluorescent signal over the cell population. It is well-known
that the rate of ROS generation upon stimulation of a cell
population is heterogeneous. 49 Therefore, as expected,
individual cells within a population exhibited different kinetics
of ROS production and therefore different detoxification
kinetics.
Conclusion. We have shown that it is feasible to develop
artificial organelles that can be taken up by cells and carry out
the function of a specific natural organelle. This represents a
milestone in the development of in vivo cell implants and a new
frontier in medical therapy. We developed artificial peroxisomes
(APs) by compartmentalizing a set of antioxidant enzymes that
act in tandem inside gated polymer vesicles. Optimization of
the vesicle properties and of the in situ cascade reaction were
necessary to achieve cell integration and to mimic the
morphology and function of a natural peroxisome. APs can
then simultaneously detect and detoxify reactive oxygen species
(ROS). By their intact escape from endolysosomes, distributing
within the cytoplasm, and being nontoxic, artificial peroxisomes
will lead to significant innovations in the therapeutics of
elevated cellular ROS and the repair of disturbed cell function
involved in the development of diseases that include cancer,
arterial sclerosis, Parkinson’s disease, and so forth. Artificial
organelles, such as our artificial peroxisomes, can be expected to
serve as cell implants and will support a major effort toward
creating artificial cells.
Methods. Preparation of APs. To produce APs, the
enzymes SOD, LPO, and CAT, or combinations thereof were
encapsulated in polymer vesicles made of PMOXA12-PDMS55PMOXA12 triblock copolymer, with outer membrane protein F
(OmpF) incorporated in the polymer vesicle membrane. The
detailed preparation technique and enzyme optimization
strategies are discussed in the Supporting Information.
Characterization of the APs. The size and morphology of
the APs were characterized by light scattering, transmission
electron microscopy (TEM), and fluorescence correlation
spectroscopy (FCS). Characterization and quantification of
the encapsulation of individual enzyme types (SOD, LPO, or
CAT) to maximize encapsulation efficiency was carried out
with FCS measurements, with the procedure described in detail
in the Supporting Information.
Co-encapsulation characterization of APs was carried out on
a Zeiss LSM 510-META/Confocor2 laser-scanning microscope
in FCCS mode, as described in the Supporting Information.
Cell Experiments. HeLa cells were cultured and maintained
in modified Eagle’s medium (DMEM) supplemented with high
glucose, 10% fetal calf serum, 1% penicillin (10 000 units/mL),
streptomycin (10 000 mg/mL) (purchased from Sigma
Aldrich), 2 mM L-glutamine, and 1 mM pyruvate. Cells were
grown in a humidified incubator (HERA Cell 150, Thermo
Scientific, Germany) at 37 °C in a 5% CO2 atmosphere until a
confluence of 60−80% was achieved and then harvested by
trypsin-ethylenediaminetetraacetic acid.
■
ASSOCIATED CONTENT
S Supporting Information
*
Detailed materials for AP preparation, detailed characterization
techniques, additional characterization results, and additional
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Nano Letters
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cell experiment information. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected].
Author Contributions
P.T. and V.B. contributed equally.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We thank Prof. W. Meier from the University of Basel for
providing the block copolymers and Dr. O. Fischer for fruitful
discussions. V.B. and P.T. acknowledge G. Persy (University of
Basel) and V. Olivieri (Microscopy Center, University of Basel)
for TEM-measurements. Authors thank M. Inglin for editing
the manuscript. Financial support was provided by the Swiss
National Science Foundation, and the National Center of
Competence in Nanoscale Science. This is gratefully acknowledged.
■
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