Synthetic bio-nanoreactor: Mechanical and chemical control of polymersome membrane permeability

Article abstract of DOI:10.1002/anie.201108814

pH-sensitive polymersomes have been stabilized by UV cross-linking after incorporation of an enzyme in a synthetic bio-nanoreactor without transmembrane proteins. This stabilization allows the reversible swelling and deswelling of the polymersomes upon changes in the pH value. The controlled reaction of the enclosed enzyme could be monitored selectively at pH 6, when the substrate could penetrate the membrane. Copyright

Full text of DOI:10.1002/anie.201108814

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DOI: 10.1002/anie.201108814
Nanoreactors
Synthetic Bio-nanoreactor: Mechanical and Chemical Control of
Polymersome Membrane Permeability**
Jens Gaitzsch, Dietmar Appelhans, Linge Wang, Giuseppe Battaglia,* and Brigitte Voit*
The combination of chemistry and biology has gained
increased interest among scientists over the past decade.
The goal of chemists has become to mimic biological
structures by chemical means.[1] A good example of this is
the development of wholly synthetic analogues of lipid
vesicles. These are thought to be critical components in the
evolution of life, since they would allow the necessary
compartmentalization for complex biochemical processes to
take place.[1,2] Within the last few years, in particular,
a polymer-based equivalent, the polymersome, has been
found to be a promising candidate for this.[3–6] Several reports
have shown that polymersomes can be used to encapsulate
Our amphiphilic block copolymer consists of well-known
biocompatible and non-immunogenic poly(ethylene glycol)
(PEG) as the hydrophilic part. The hydrophobic part is
a statistical copolymer of the pH-sensitive diethyl amino ethyl
methacrylate (DEAEM) and a photo-cross-linking unit of
either 3,4-dimethyl maleic imidoethyl methacrylate
(DMIEM, C2 polymers) or 3,4-dimethyl maleic imidobutyl
methacrylate (DMIBM, C4 polymers) in 10 or 20 mol%,
respectively (Scheme 1 and Table 1).
We showed previously[22] that our copolymers C2-10 and
C2-20 (Scheme 1) are able to form polymersomes with
a photo-cross-linkable and pH-sensitive moiety. Furthermore,
the transmembrane traffic of dye molecules through cross-
linked polymersomes could be triggered by changes in the
pH value.[22] However, the cross-linking time of 80 minutes[22]
was likely to destroy any functionality of a bioactive molecule
eventually enclosed within. We are now able to reduce the
time of the UV irradiation necessary to reach a cross-linked
DNA,[7] RNA,[8,9] as well as functional enzymes.[4,5,10–14]
A
great advantage of polymersomes over liposomes is their
higher mechanical and chemical stability. The high flexibility
in the chemical design of polymersomes allows the formation
of membranes with tunable permeability.[15–19] This approach
can be further enhanced by the incorporation of active
molecular transporters such as transmembrane proteins.[5,20]
However, it is not certain whether polymersome membranes
can withstand high shear rates, which may occur during an
industrial cleaning process.[16] Here, we present a new
approach to modulate the permeability of polymersome
membranes by using pH-sensitive photo-cross-linkable
copolymers. We aimed to generate cross-linked polymer-
somes to tune the shear-rate-induced release of globular
dendritic glycopolymers[21] of different sizes. The concept was
then further expanded to an enzymatic reaction within
polymersomes, with the aim of controlling transmembrane
traffic by modulating the pH value.
Scheme 1. a) Chemical structure of amphiphilic block copolymers with
an ethyl (C2) and butyl (C4) spacer in the cross-linking units DMIEM
and DMIBM, which are used in 10 or 20 mol%, respectively (resulting
in C2-10, C2-20, C4-10, and C4-20 nomenclature) and b) the cross-
linking reaction occurring within the membrane.
[*] J. Gaitzsch, Dr. D. Appelhans, Prof. B. Voit
Leibniz-Institut fꢀr Polymerforschung Dresden e.V.
Hohe Strasse 6, 01069 Dresden (Germany)
J. Gaitzsch, Prof. B. Voit
Organische Chemie der Polymere
Fachrichtung Chemie und Lebensmittelchemie
Technische Universitꢁt Dresden
01062 Dresden (Germany)
Table 1: Properties of the polymers and polymersomes (Psome),
including cross-linking time of the polymersomes.
[a]
Mn
Cross-linker[b]
(mol%)[c]
P’some size[d]
PDI[d]
t[e]
[s]
E-mail: voit@ipfdd.de
[kgmolÀ1
]
[nm]
Dr. L. Wang, Prof. G. Battaglia
C2-10
C2-20
C4-10
C4-20
19.0
21.5
20.0
22.5
C2 (10)
C2 (20)
C4 (10)
C4 (20)
125
120
100
125
0.2
0.2
0.2
0.2
180
120
120
30
Department of Biomedical Science, University of Sheffield
Firth Court, Western Bank, Sheffield S10 2TN (UK)
E-mail: g.battaglia@sheffield.ac.uk
[**] Financial Support from the Rosa-Luxemburg-Foundation and the
Dresden International Graduate School for Biomedicine and
Bioengineering (DIGS-BB) is gratefully acknowledged.
[a] Mn is determined from signal intensities in the 1H NMR spectra; the
PDI of the copolymers is 1.3 (1.4 for C4-10). [b] Refers to the length of the
carbon chain (Scheme 1a). [c] With respect to the hydrophobic block
length; determined from signal ratios in the H NMR spectra.
[d] Determined by DLS (diameter), PDI=polydispersity of polymer-
Supporting information for this article (experimental section and
data on the polymersomes, release experiments, and enzymatic
reaction) is available on the WWW under http://dx.doi.org/10.
1002/anie.201108814.
1
somes. [e] Cross-linking time.
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state from over 60 minutes to only 30 seconds. This has been
achieved by changing the UV source, and also by using
a cross-linking unit with a longer spacer. The polymer series
C2-10, C2-20, C4-10, and C4-20 were prepared, and the cross-
linked polymersomes made up of the C4-20 polymer required
only 30 seconds of UV irradiation (Table 1). The cross-linking
unit in C4 polymers is attached to a butyl spacer (PDMIBM)
instead of the ethyl spacer (PDMIEM) in the C2 polymers
(Scheme 1). We postulate that less steric hindrance between
the PDMIBM and neighboring PDEAEM molecules leads to
a higher cross-linking efficiency. The C4-20 polymer was used
for further studies because it required the shortest cross-
linking time to form the polymersomes.
The next steps were to determine the stability of the cross-
linked polymersomes at various pH values. Transmission
electron microscopy (TEM) and dynamic light scattering
(DLS) studies showed that the cross-linking of the membrane
hinders the disassembly of the polymersomes of the C4-20
polymer at acidic pH values (Figure 1a,c). In contrast, in non-
cross-linked polymersomes, C4-20 is fully hydrophilic at an
Figure 2. Pressure-dependent release of fluorescein-labeled PEI-Mal 25
and PEI-Mal 5 from cross-linked and non-cross-linked polymersomes
at pH 4 and 9. The retention of the nanoparticles within the polymer-
somes was observed by the absorption maxima of fluorescein at
500 nm. TMP=transmembrane pressure.
some membrane, as measured by TEM, that matches the
increase in the vesicle diameter determined by DLS studies
(Figure 1).
To determine the mechanical stability and permeability of
our pH-sensitive polymersomes in a cross-linked and non-
cross-linked state (Figure 2) we studied the effect of the shear
rate on polymersomes containing maltose-decorated hyper-
branched polyethyleneimine (PEI-Mal) nanoparticles.[21] PEI
cores of 5000 Da (PEI-Mal 5, diameter 4 nm) and 25000 Da
(PEI-Mal 25, diameter 10 nm) were used to study their
release at various pH values and shear rates. These readily
available fluorescein-labeled organic nanoparticles are pro-
tein-sized and softer than traditional silica or gold nano-
particles, thus making them ideal test molecules. They were
enclosed within the polymersomes during the self-assembly of
C4-20, and then the vesicles were exposed to different
conditions (Figure 2).
As a starting point, we demonstrated that both dendritic
nanoparticles can be encapsulated within non-cross-linked
polymersomes and retained after purification by dialysis at
pH 9 (Figure 2). However, dialysis with classical dialysis tubes
is a time-consuming process that takes several days. More-
over, this technique has the drawback that no shear rate can
be applied during the polymersome cleaning process. The
application of a pressure to the solution would speed up
various processes of interest by placing polymersomes under
different shear rates.
Figure 1. Development of the polymersomes upon changes in the
pH value. a) Reversible change in the diameter of polymersomes
between pH 3 and pH 10 of the C4-20 polymer, as measured by DLS.
b) The membrane thickness changes in the same proportion as the
vesicles. c,d) Vesicles (sketches shown) could be visualized at pH 3 (c)
and pH 10 (d) by using TEM.
acidic pH value and hence no longer able to form polymer-
somes (Figure 2). Furthermore, cross-linked polymersomes
show a definite swelling/deswelling cycle upon switching of
the pH value, which can be repeated at least five times
(Figure 1a, and Figure 4-SI in the Supporting Information).
This cycling is entirely due to the physicochemical changes of
the PDEAEM chains in going from an unprotonated, hydro-
phobic entangled state at a high pH value to a protonated,
hydrated, hydrogel-like state at a low pH value. Such a change
corresponds with an increase in the thickness of the polymer-
Consequently, we tested the purification process using
a KrosFlo Research IIi system (also known as a hollow fiber
filtration (HFF) system) and applying transmembrane pres-
sures (TMP) of 250 and 750 mbar (Figure 2). The time for the
purification of the polymersomes could be shortened to
1 hour at 250 mbar TMP and to only 30 min at 750 mbar TMP.
In a first experiment, non-cross-linked polymersomes hosting
the two different nanoparticles PEI-Mal 5 and PEI-Mal 25
were processed at 250 mbar and pH 9. Here, only the large
nanoparticles (PEI-Mal 25) could be retained, but not PEI-
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Mal 5. This result indicates that some polymersomes rupture
as a consequence of the shear rate and undergo cargo loss. We
assume this rupture occurs through the formation of small
pores (i.e. poration) within the membrane that heal over after
the shear rate is removed. Furthermore, this membrane
poration is accompanied by a polymersome squeezing/
deforming process when pressure is applied. DLS studies
carried out after each shear-rate experiment confirmed that
the polymersomes are still detectable (see the Supporting
Information).
The finding that neither type of PEI-Mal nanoparticles
are retained at a TMP of 750 mbar at pH 9 when using non-
cross-linked polymersomes indicates that the pore size
depends on the shear rate (see Figure 2 and the Supporting
Information). We performed the same process at a TMP of
750 mbar and pH 9 with cross-linked polymersomes. While
the small PEI-Mal 5 nanoparticles still exit the polymersomes,
the larger PEI-Mal 25 nanoparticles could now be detected
after the procedure (Figure 2). This observation suggests that
membrane cross-linking increases the resistance to shear-
rate-induced poration compared to non-cross-linked mem-
branes. The polymersome membrane still retains its charac-
teristic ability to form pores and self-heal (see the Supporting
Information). Neither nanoparticles are retained when swol-
len, cross-linked polymersomes are processed, for example, in
acidic media (pH 4) and at a low TMP of 250 mbar. Hence, we
can conclude that polymersomes (whether their membrane is
cross-linked or not) respond to an applied shear rate by
forming controllable, definite-sized pores (see Figure 2 and
the Supporting Information). The use of such an approach
would thus enable us to design polymersomes whose perme-
ability could be finely controlled by the shear rate as well as
the cross-linking state and the solution pH value.
Figure 3. Schematic representation of the reaction of guaiacol and
hydrogen peroxide in myoglobin-filled polymersomes: First at a basic
(b), then at an acidic (b-a), and again at a basic (b-a-b) pH value.
Hydrogen peroxide is added 120 s after the addition of guaiacol. The
graph shows the normalized time-dependent absorption at 470 nm.
To further validate the responsive nature of the cross-
linked polymersomes we created a bionanoreactor by incor-
porating myoglobin as a model enzyme. This is a very well
characterized enzyme[23] with an iron-containing central
complex. Myoglobin is able to catalyze oxidative reactions,
for example, the well-known reaction of guaiacol with
hydrogen peroxide. An advantage of this enzymatically
controlled reaction is that the final product (oxidised
guaiacol) has an absorption maximum at 470 nm and thus
UV/Vis spectroscopy can be used to monitor the reaction.[23]
We first tested the ability of myoglobin to withstand the UV
irradiation necessary for cross-linking the polymersomes. In
the absence of polymersomes, the enzyme still shows high
catalytic activity after irradiation (see the Supporting Infor-
mation). After incorporating myoglobin within the polymer-
somes and applying our shear-rate-induced purification step
(200 mbar TMP), we cross-linked the polymersomes and then
checked the activity of the enclosed enzyme under various
conditions (Figure 3).
finding clearly indicates that the reagents cannot diffuse into
the polymersome lumen where the enzyme is localized. In
contrast, a higher and longer increase in the absorption of the
reaction product, indicating a catalyzed reaction between
guaiacol and hydrogen peroxide within the bionanoreactor,
was evident at pH 6 (Figure 3, “b-a” state). The polymersome
membrane is now in a swollen state and totally hydrophilic.
Transmembrane diffusion is thus possible, thereby allowing
the guaiacol and hydrogen peroxide to reach the myoglobin,
which catalyzes the reaction. As the pH value is increased
again to 8, the reaction is stopped because the nonpermeable
polymersome membrane reforms (Figure 3, “b-a-b” state; for
further details see the Supporting Information). Thus, the
myoglobin inside the polymersomes is no longer accessible to
the reagents from outside. This also indicates that myoglobin
cannot diffuse out of the polymersomes if no shear-rate
conditions are applied.
In conclusion, we have successfully demonstrated that
polymersomes undergo selective shear-rate-induced release
of two different nanometer-sized dendritic glycopolymers
through controlled poration of the membrane. These mem-
brane pores can be controlled by the shear rate, cross-linking,
and the pH value. This specific approach for shear-rate-
induced release is only possible with our pH-sensitive, photo-
cross-linkable polymersomes. We showed the simultaneous
The first experiments showed that guaiacol does not react
in the absence of hydrogen peroxide, as determined by
measuring the myoglobin activity at pH 8. At this pH value,
the PDEAEM polymersome membrane is unprotonated and,
therefore, fully hydrophobic. Consequently, no enzymatic
activity was observed after adding hydrogen peroxide to the
bionanoreactor solution at pH 8 (Figure 3, “b” state). This
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the need to use a transmembrane protein to control the feed
of an enzyme into the polymersome lumen. The pore size and
transmembrane trafficking of the reagents can be controlled
in the case of enzymatic reactions inside polymersomes by
pH-induced swelling and shrinking of the pH-stable poly-
mersomes.
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Received: December 14, 2011
Published online: March 21, 2012
Keywords: bio-nanoreactors · membranes · polymersomes ·
.
poration · shear force
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