In situ ATRP-mediated hierarchical formation of giant amphiphile bionanoreactors

Article abstract of DOI:10.1002/anie.200801007

(Figure Presented) Functional giants: Amphiphilic bioconjugates can be synthesized in situ by grafting polystyrene from a protein (see scheme). The resulting giant amphiphiles display low polydispersities and the characteristic aggregation properties of amphiphilic biomacromolecules. A second, catalytically active guest protein can also be included within the superstructures.

Full text of DOI:10.1002/anie.200801007

Angewandte
Chemie
DOI: 10.1002/anie.200801007
Bionanoreactors
In Situ ATRP-Mediated Hierarchical Formation of Giant Amphiphile
Bionanoreactors**
Benjamin Le Droumaguet and Kelly Velonia*
Hierarchical architectures with a broad range of lengths are
ubiquitous in biological material systems. In nature, most of
these multifunctional materials are produced in situ under
ambient conditions and through ecologically balanced pro-
cesses. Synthesized to mimic natural superstructures, giant
successful application of ATRP for the formation of giant
amphiphiles would offer an efficient synthetic alternative, but
more importantly it would allow for the first time their
hierarchical programming into the construction of multi-
enzyme superstructures.
We report here on the facile and high-yielding ATRP-
mediated preparation of giant amphiphiles in situ (Scheme 1).
We prove that by utilizing ATRP, quantitative amounts of
[
1–3]
[4–10]
amphiphiles,
a subclass of protein–polymer conjugates
in which the hydrophobicity of the polymer conveys overall
amphiphilic character to the biohybrid, exhibit interesting
aggregation properties and enormous potential in bio- and
nanotechnology.
In the past, these bioconjugates have been prepared either
through the direct conjugation of appropriately functional-
[
1]
[3]
ized macromolecules to specific amino acids or cofactors,
[2]
or through bioaffinity couplings. The efficiency of their
synthesis by these conventional pathways was hampered by
practical limitations caused mainly by the solubility incom-
patibility between the protein and the hydrophobic polymer
and by constraints posed to guarantee the stability of the
protein itself. Moreover, concomitant to their synthesis, stable
nondynamic superstructures formed, accounting for the
tedious purification required and the low yields; this signifi-
cantly limited any further exploration of the potential of giant
amphiphiles in the creation of preprogrammed multifunc-
tional systems.
To overcome the intrinsic limitations caused by either the
synthetic approaches or the amphiphilic nature of such
bioconjugates, we employed protein-initiated atom-transfer
radical-mediated polymerization (ATRP), a method recently
[
11,12]
introduced for the preparation of hydrophilic
and
[11]
smart polymer–protein conjugates. We envisioned that a
[
+]
[*] B. Le Droumaguet, Dr. K. Velonia
Department of Chemistry, University of Geneva
Quai Ernest Ansermet 30, Sciences II
CH 1211 Geneva 4 (Switzerland)
Scheme 1. General scheme for the in situ ATRP-mediated synthesis of
giant amphiphiles.
+
[
] Present address:
Department of Materials Science andTechnology
University of Crete
University Campus Voutes, 71003 Heraklion, Crete (Greece)
http://www.materials.uoc.gr/proswpiko/velonia.htm
Fax: (+30)2810-394-273
bioconjugates possessing low polydispersities and the char-
acteristic aggregation properties of amphiphilic biomacro-
molecules form. More importantly, we demonstrate for the
first time the construction of hierarchically assembled bio-
nanoreactors by the efficient one-pot incorporation of a guest
protein within the formed superstructures.
E-mail: velonia@materials.uoc.gr
[
**] The authors acknowledge the Swiss National Science Foundation
(
200020-113804/1) for financial support. We thank D. Gerardfor the
The efficiency of in situ protein-initiated ATRP for the
synthesis of giant amphiphiles was explored using the 66 kDa
globular bovine serum albumin (BSA), which contains only
synthesis of the initiator, Dr. C. Bauer andJ. Bosset at the
Bioimaging Platform of the University of Geneva for their assistance
with microscopy, andD.-H. Tran andthe SVSMS Mass Spectrometry
Core Facility of the University of Geneva for the MALDI-TOF MS
measurements.
[
13]
one free cysteine residue at position 34.
Adapting the
approach used by Maynard et al. for the formation of smart
[
11]
protein conjugates, we synthesized the maleimido-capped
Supporting information (including full descriptions of all experi-
ments conducted in this study) for this article is available on the
WWW under http://dx.doi.org/10.1002/anie.200801007.
[
14]
ATRP initiator I (Scheme 1).
Its subsequent bioconju-
gation reaction was performed under mild conditions by the
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slow addition of a solution of I (40 molequiv) in DMSO (4%
position (see Table S1 in the Supporting Information). In all
cases, the resulting bioconjugate solutions were subjected to
extensive dialysis to ensure that all traces of reagents (styrene,
copper, N-(n-propyl)-2-pyridylmethanimine) were removed.
When low monomer/II ratios were employed (50:1 and
500:1), SEC revealed mixtures of unreacted II and biocon-
jugates III with a hydrodynamic volume slightly higher than
that of BSA (Figure 1). Native gel electrophoresis revealed a
band of lower electrophoretic mobility than that of BSA
(higher molecular weight). MALDI-TOF spectrometry veri-
fied these findings through m/z signals corresponding to
unreacted II, along with signals ranging from 67 to 71 kDa for
III. These reactions were accompanied by low yields (full data
in the Supporting Information).
When high styrene/II ratios were utilized (1500:1 to
3000:1), SEC analysis revealed the quantitative formation of
giant amphiphiles III having a larger hydrodynamic volume
than BSA (Figure 1A). In all cases, RI (RI = refractive index)
and UV traces were in good agreement. The broadness of the
peaks observed for BSA, the macroinitiator II, and the giant
amphiphiles III was similar, indicating both the efficiency of
the polymerization and the retention of dispersity. Native gel
electrophoresis revealed behavior typical for giant amphi-
philes, that is, migration hampered by the amphiphilic
character of the bioconjugate and no trace of the starting
materials. MALDI-TOF analyses showed m/z signals ranging
from 71 to almost 80 kDa depending on the monomer/II ratio
utilized (Figure 1B) and no signal corresponding to the
macroinitiator II. The TEM images verified the in situ
formation of spherical aggregates with behavior similar to
final volume content) to BSA. The reaction mixture was
overincubated (48 h at 78C) for maximum yields, and the
bioconjugate II was easily purified by extensive dialysis.
The quantification of the reaction with Ellmanꢀs colori-
[15]
metric assay revealed practically no free conjugation sites
Cys) in the product solution; native gel electrophoresis
(
revealed a new single band for II; and size-exclusion
chromatographic (SEC) analyses showed a peak with same
broadness as and a retention time only slightly different from
that of native BSA. No trace for the free ATRP initiator I was
detected upon extensive dialysis. The quantitative formation
of II was verified by MALDI-TOF analyses, which revealed
peaks at masses of 66313 and 66737 amu for native BSA and
the BSA–macroinitiator II, respectively, a shift corresponding
to the addition of one molecule of maleimido initiator I per
protein (Figures S1–S3in the Supporting Information).
The monomer employed for the subsequent polymeri-
zation was styrene, as it would lead to polystyrene giant
amphiphiles known to exhibit a strong amphiphilic charac-
[
1]
ter. ATRP on II was performed in aqueous solution, in the
absence and presence of DMSO as cosolvent, under oxygen-
free conditions, at ambient temperature, with the copper
bromide/N-(n-propyl)-2-pyridylmethanimine
catalyst
[16]
[11]
system, and without any “sacrificial” initiator. Several
sets of experiments were performed utilizing different ratios
monomer to biomacroinitiator II, while a series of control
I
experiments (in the absence of II or styrene or Cu , and in the
presence of O ) were conducted to ensure that the in situ
2
polymerization proceeds on the designated BSA initiating
Figure 1. Characterization of the BSA–polystyrene biohybrids synthesized utilizing ATRP and high monomer/II ratios. Representative A) SEC
traces, B) MALDI-TOF spectra, andC) TEM images. D) MALDI-TOF spectrum of isolatedpolystyrene.
6
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Angewandte
Chemie
[
16b]
that of conventionally synthesized giant amphiphiles
and
diameters ranging from 20 to 100 nm (Figure 1C; see the
Supporting Information).
To further prove the formation of polystyrene, the
biopolymers III were subjected to HCl-mediated protein
degradation. The structure of the isolated polymer samples
was verified by NMR spectroscopy and MALDI-TOF
spectrometry (full data in the Supporting Information). It is
worth noting that the MALDI-TOF spectrum revealed only
two major distributions of the isolated polymer (Figure 1D),
one of which was predominant and displayed a rather low
polydispersity index in comparison to that of a standard
polystyrene of known polydispersity. We attribute this low
polydispersity to the selected synthetic approach.
Finally, this type of “grafting from” by means of the ATRP
technique was successfully applied to two other proteins,
namely, human serum albumin (HSA) and reduced human
calcitonin, thus demonstrating the generality of this method
for the formation of giant amphiphiles. (Full experimental
details are included in the Supporting Information.)
We confirmed the nature of the in situ generated giant
amphiphiles but more significantly their capacity to concur-
rently form hierarchically assembled nanocontainers by
performing the polymerization reaction in the presence of a
second nonpolymerizable protein. For this reason, fluores-
cently labeled papain or horseradish peroxidase (HRP) was
integrated into the optimized reaction scheme (i.e. high
styrene/II ratios) and a supplementary final dialysis step was
added to remove the non-encapsulated proteins. In both
cases, aliquots from the reaction mixtures and purified
solutions were analyzed by SEC, which revealed the forma-
tion of the nanocontainers and the elimination of the non-
encapsulated proteins by dialysis. The TEM micrographs
Figure 2. Hierarchically formednanocontainers. A) CFM images and
B) TEM images of papain-loaded nanocontainers labeled with fluores-
cein andAtto. C) Activity profile of HRP-loa de dnanoreactors.
eliminated. We proved that by the creation of such chimeric
systems in situ, the one-pot hierarchical incorporation of
other species (such as enzymes) is possible without steps that
would interfere with the protein integrity or the overall
architecture of the aggregate. Furthermore, we demonstrated
that these novel giant amphiphile nanocontainers are perme-
able and can be used as nanoreactors. Our current efforts are
focused on the full exploitation of the ATRP technique for
the preparation of functional polymer–biomolecule conju-
gates and multienzyme nanoreactors.
(
Figure 2B) showed aggregation patterns similar to those
observed for the polymerizations of II in the absence of a
second protein.
The presence of the fluorescently labeled papain within
the spherical superstructures was demonstrated by confocal
fluorescence microscopy (CFM). When these nanocontainers
were subjected to a second, external labeling with Atto, CFM
revealed the expected statistical presence of both fluorescent
species in the superstructures (Figure 2A).
The catalytic efficiency and permeability of these hier-
archically formed nanocontainers was tested using a solution
of the purified biohybrid III loaded with HRP and by
Received: March 2, 2008
Published online: July 10, 2008
Keywords: bioconjugates · hierarchical assembly ·
.
nanostructures · polymerization
employing the standard TMB/H O2 (TBM = 3,3’,5,5’-tetra-
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