Monodisperse polymer-virus hybrid nanoparticles

Article abstract of DOI:10.1039/b613890j

The interaction of polystyrene sulfonate with coat proteins (CP) at neutral pH in different stoichiometric conditions and the formation of monodisperse nanoparticles were investigated. The nanoparticles were characterized using fast performance liquid chr

Full text of DOI:10.1039/b613890j

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Monodisperse polymer–virus hybrid nanoparticles†
Friso D. Sikkema,a Marta Comellas-Aragone`s,a Remco G. Fokkink,b Benedictus J. M. Verduin,c
Jeroen J. L. M. Cornelissen*a and Roeland J. M. Noltea
Received 25th September 2006, Accepted 10th November 2006
First published as an Advance Article on the web 17th November 2006
DOI: 10.1039/b613890j
Self-assembly of polystyrene sulfonate and modified cowpea
chlorotic mottle virus protein yields monodisperse icosahe-
dral nanoparticles of 16 nm size.
(7.5) and dialyzed to pH 4.5 give rise to particles corresponding
to the dimensions of the virus (T = 3).7 No well-defined particles
are observed at high pH. Incubation of the CP at pH 7.5 with ds-
DNA of different sizes has been shown to yield 16–17 nm diameter
tubular structures of various lengths.8 This implies that at low pH
(i.e. 5), interactions between protein subunits predominate capsid
formation, while at higher pH (≥7) other interactions (such as
between CP and polyelectrolyte) become increasingly important.
We have investigated the interaction of polystyrene sulfonate
(average mass: 9900 Da) with CP at neutral pH in different stoi-
chiometric conditions and report here the formation of monodis-
perse 16 nm icosahedral T = 1 nanoparticles, characterized using
fast performance liquid chromatography (FPLC), transmission
electron microscopy (TEM) and dynamic light scattering (DLS).
Organic and inorganic nanoparticles are currently receiving great
interest because of their interesting physical and chemical proper-
ties, which are often different from those of microscopic systems.1
These have been synthesized by a wide variety of methods, one
being the self-assembly of amphiphilic block-copolymers, which
yields vesicles, micelles and other structures. The use of biological
materials to create nanoparticles is increasing.2 Many efforts
have been directed toward the encapsulation of drugs and other
molecules in these particles.3 Major disadvantages, however, are
their low biocompatibility, their low solubility in water and in
general their broad polydispersity, although small polydispersity
indices have been attained.4
The well-characterized virus cowpea chlorotic mottle virus
(CCMV) consists of 90 identical dimeric coat proteins (CP)
enveloping a central RNA strand, yielding a highly defined 28 nm
virion.5 The icosahedral shape of the virus can be described
according to the Caspar–Klug T (triangulation) number.6 A larger
T number describes a larger particle, with a set number of protein
subunits. In CCMV, the 90 dimeric coat protein subunits give rise
to a T = 3 particle, consisting of 12 pentameric arrangements
and 20 hexameric faces. The monomeric 20 kDa CP consists of
189 amino acids with nine basic residues at the N-terminus. In
its free form, the CP is dimeric. Ionic interactions between the
RNA and these residues enhance the structural integrity of the
virus, so that capsid assembly at neutral pH is only possible in
the presence of RNA.6 Removal of the RNA by precipitation
yields pure CP which can be assembled by lowering the pH to
∼5, yielding empty T = 3 capsids. Very little or no empty capsid
will form in neutral conditions (see below). It is believed that
subtle electrostatic interactions between the RNA and the protein
subunits assist in particle formation. Experiments to determine
if a synthetic polymer gives rise to similar effects have been
undertaken: It has been reported in literature that negatively
charged sodium–polyanethole sulfonate and CP mixed at high pH
CP was prepared according to
a
literature procedure.9
Polystyrene sulfonate was synthesised using the atom transfer
radical polymerization (ATRP) technique.10 In order to facilitate
detection by UV, the polymer was equipped with a dansyl group.
To achieve this, dansyl chloride (Dns-Cl) was first reacted with
1,3-diaminopropane (Scheme 1) to yield 1, which was coupled
to 1-bromoethyl benzoic acid to provide the ATRP initiator 2.
Monomer 3 was prepared using a modified literature procedure.11
Subsequent polymerization of 3 in the presence of 2, CuBr and
2,2ꢀ-bipyridine yielded the desired polymer, DNs-PSS (4) after
deprotection of the ethyl ester functions.
A fixed amount of CP (4.5 mg ml−1) in a pH 7.5 Tris buffer
(50 mM, 0.3 M NaCl) was incubated with varying amounts of
DNs-PSS and the assembly of the particles was monitored by
FPLC and TEM.12 Fig. 1 shows the FPLC elution curves for CP,
incubated with different amounts of DNs-PSS.
The incubation time appeared not to be a crucial parameter
since no difference was detected between a sample injected 1 hour
after incubation and one that had stood at 4 ◦C for several days.
Thepresence of polymer was verifiedby UV(adsorptionat312nm,
see the ESI†). A very small ratio of DNs-PSS : CP (0.4)13 yielded
very little particle assembly, with small peaks at elution volumes
of 1.0 and 1.35 ml, corresponding to T = 3 icosahedral particles
(the naturally occurring capsid of 180 subunits) and non-natural,
somewhat smaller particles. Increasing the ratio of DNs-PSS : CP
to 4 and then to 40 caused the peak at 1.0 ml to decrease and the
one at 1.35 ml to increase. At the ratio of 40 only the assembly
eluting at 1.3 ml and free dimeric CP was detected, implying that
this ratio is close to the ideal value for formation of this particular
assembly. Increasing the ratio DNs-PSS : CP even more (400)
yielded these particles as well, but the FPLC trace clearly showed
a lower intensity of the 1.35 mL peak, and a large amount of
material eluting at 1.6 ml (DNs-PSS). This inhibition of particle
formation is probably caused by a polyelectrolyte effect, preventing
capsid formation at high polyelectrolyte concentrations. It is, for
aInstitute for Molecules and Materials, Radboud University Nij-
megen, Toernooiveld 1, 6525 ED, Nijmegen, the Netherlands. E-mail:
J.Cornelissen@science.ru.nl; Fax: +31 (0)24 365 2929; Tel: +31
(0)24 365 2381
bLaboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD,
Wageningen
cLaboratory of Physical Chemistry and Colloid Science, Wageningen
University, Dreijenplein 6, 6703 HB, Wageningen
† Electronic supplementary information (ESI) available: Synthesis, ex-
perimental procedures and detailed inclusion experiments. See DOI:
10.1039/b613890j
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Scheme 1 Synthesis of labelled polystyrene sulfonate (DNs-PSS). For synthetic details see the ESI.†
Table 1 Relative peak intensities in the FPLC chromatograms
Relative peak intensitya
Ratio DNs-PSS : CP
T = 3b
T = 1c
CPd
0.4
4
40
4
3
0
0
6
48
68
37
90
48
32
63
400
a In% based on the peak heights in the FPLC chromatogram detected at
k = 208 nm. b Eluting at v = 1.0 ml. c Eluting at v = 1.35 ml. d Eluting at
v = 1.8 ml.
Fig. 1 FPLC traces of mixtures containing different ratios of DNs-PSS
polymer and CP subunits The resolution of the FPLC systems does not
allow discrimination between native and degraded CP (see below). Small
ratios of DNs-PSS : CP (0.4 and 4)13 induce formation of two types of
particles, with elution volumes of 1.0 and 1.35 ml, indicating the presence
of T = 3 and T = 1 protein aggregates. At a ratio of 40 only T = 1 particles
and free CP appear. Higher ratios of DNs-PSS/CP (400) inhibit particle
formation.
example, possible that at higher concentrations multiple polymer
chains engage in an interaction with CP so that particle formation
is inhibited. Since no peak appears for the polymer, not even at
high magnification of the traces, we may conclude that, at lower
stoichometries, all polymer is encapsulated within the particles.
Blank FPLC runs with only polymer (not shown) did not show a
significant change in elution volume (∼1.55 ml) as a function of
concentration, implying that free DNs-PSS is always detected at
the same elution volume.
Fig. 2 Transmission electron micrograph of T = 1 assemblies of CP
assembled around DnS-PSS. The scale bar represents 100 nm. Size analysis
of the particles revealed that they have a diameter of 16.1 to 16.2 nm.
The peak intensity of a given elution volume, relative to total
protein and polymer peak intensity was calculated (Table 1). For
the T = 1 particle, indeed the highest relative peak intensity was
found at the ratio DNs-PSS : CP = 40.
stable; storage at 4 ◦C for many weeks had no detectable effect on
the particles.
Fig. 3 shows the results of dynamic light scattering experiments
of the isolated aggregate, which clearly indicates that the new T =
1 particle is smaller (R ∼ 9.5 nm) than unmodified CCMV. The dis-
crepancy between the observed diameter in the TEM micrograph
TEM images (Fig. 2) obtained from the fraction collected
at 1.35 ml revealed the presence of well-defined monodisperse
particles which were around 16 nm in diameter and remarkably
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Fig. 3 Normalized CONTIN fits of dynamic light scattering data for the CCMV T = 3 particle and the DnS-PSS : CP T = 1 aggregate.
and the radius obtained from DLS measurements is likely caused
by the fact that DLS measures the hydrodynamic radius and
TEM does not. The FPLC fractions containing the DnS-PSS :
CP aggregate (pH 7.5) were reinjected on the FPLC column, and
also dialysed to pH 5.0 to verify the influence of pH change. Both
at pH 7.5 and at pH 5.0, the same particles were detected, and no
free CP was observed, implying high particle stability.
The spontaneous formation of the much smaller T = 1 particle,
compared to the native-like T = 3 capsid, was initially quite
surprising as it is well-established that native CP is unable to
form the T = 1 shell.14 Sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) analysis of the FPLC fraction corre-
sponding to the T = 1 particle indicated that the polymer loaded
particles contained modified capsid proteins lacking the (1–25) and
(1–41–44) amino acid regions (Fig. 4). SDS-PAGE of the CP used
for these experiments showed identical degradation products (not
shown). Recent results from Johnson and coworkers14 show that
purified partially degraded protein building blocks (ND34) will
form a mixture of T = 1,2,3 particles at low pH. They conclude
that the N-terminal amino acid residues 1–36 are not essential
for capsid formation but bias the T = 3 capsid arrangement.
Despite the elimination of the N-terminal RNA binding sequence,
there are still sufficient RNA binding sites left to account for the
favorable interaction between the modified CP and the polyanion.5
We speculate that the presence of a small quantity of non-degraded
protein would not exert a large influence, since our work shows that
particle formation is primarily driven by the presence of polyanion.
Nevertheless, we are currently investigating the relation between
protein degradation and particle formation.
Fig. 4 SDS PAGE analysis of the isolated T = 1 fraction (lane 1), crude
CP (lane 2) (the CP used for the inclusion experiments showed similar
degradation products as seen in lane 1) and the purified virus (lane 3). The
arrows indicate the degraded proteins lacking the (1–25) (arrow a) and the
(1–41–44) (arrow b) regions.
are formed, suggesting that in this case, the interactions between
the CP itself determine the particle size.14 We conclude that at
pH 7.5 an equilibrium exists between free native and degraded
CP and the T = 3 capsid, although strongly in favor of the free
CP. Apparently, the very small amount of polyanion present has
The experiments presented above show that at very low polymer
concentrations (DNs-PSS : CP = 0.4 and 4) larger T = 3 particles
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only a slight ionic interaction with the degraded CP, which is
not strong enough to completely change the particle dimension,
therefore, both the T = 1 and the T = 3 particles can be obtained.
In this case, the equilibrium between free CP and the T = 3 particle
still plays a role (Fig. 5). The absence of the pseudo T = ‘2’ particle
in our experiments is in line with the dynamic character of this
process, since this D = 25 nm particle is a kinetic product only
observed upon swift lowering of the pH.14
induce the formation of larger than T = 1 assemblies, opening
the way to specifically tune the particle sizes. This is currently
under investigation. Potential applications of these materials can
be found in pharmaceutics (e.g. targeting and transport), nano-
arrays or electronics.15
Acknowledgements
This work was supported by the Chemical Council of the
Netherlands Organization for Scientific Research (NWO-VW) for
a TOP grant to R. J. M. N. and a Vidi grant to J. J. L. M. C. and by
the Royal Netherlands Academy for Arts and Sciences (KNAW).
References
Fig. 5 The presence of DNs-PSS induces the formation of the T = 1
particle from modified CP (left) at pH = 7.5, whereas T = 1,2,3 particles
are formed at pH = 5.0 in the absence of polymer.
1 R. A. van Delden, M. K. J. ter Wiel, M. M. Pollard, J. Vicario, N.
Koumura and B. L. Feringa, Nature, 2005, 437, 1337–1340.
2 See for example: (a) D. M. Vriezema, M. Comellas-Aragone´s, J. A. A. W.
Elemans, J. J. L. M. Cornelissen, A. E. Rowan and R. J. M. Nolte,
Chem. Rev., 2005, 105, 1445–1489; (b) Z. Kanaras, Wang, A. D. Bates,
R. Cosstick and M. Brust, Angew. Chem., Int. Ed., 2003, 42, 191–194;
(c) X. Xu, N. L. Rosi, Y. Wang, F. Huo and C. A. Mirkin, J. Am. Chem.
Soc., 2006, 128, 9286–9287.
3 L. Mu and S. S. Feng, J. Controlled Release, 2003, 86, 33–48.
4 (a) J. Park, E. Lee, N.-M. Hwang, M. Kang, S. C. Kim, Y. Hwang, J.-G.
Park, H.-J. Noh, J.-Y. Kim, J.-H. Park and T. Hyeon, Angew. Chem.,
Int. Ed., 2005, 44, 2872–2877; (b) H. J. Hah, J. S. Kim, B. J. Jeon, S. M.
Koo and Y. Eun, Chem. Commun., 2003, 1712–1713.
5 (a) J. B. Bancroft, C. E. Bracker and G. W. Wagner, Virology, 1969,
23, 324–35; (b) J. A. Speir, S. Munshi, G. Wang, T. S. Baker and J. E.
Johnson, Structure, 1995, 3, 63–78.
6 L. O. Liepold, J. Revis, M. Allen, L. Oltrogge, M. Young and T. Douglas,
Phys. Biol., 2005, 2, S166–S172 and references cited.
7 T. Douglas and M. Young, Nature, 1996, 393, 152–155.
8 (a) J. B. Bancroft, E. Hiebert and C. E. Bracker, Virology, 1969, 39,
924–930; (b) S. Mukherjee, C. M. Pfeifer, J. M. Johnson, J. Liu and A.
Zlotnick, J. Am. Chem. Soc., 2006, 128, 2538–2539.
At higher stoichiometric ratios, the presence of more attractive
interactions force the modified CP to aggregate in a T = 1
architecture, which was found to be remarkably stable; equilibria
existing between this T = 1 particle and the free CP mixture or
other particles are strongly in favor of the former species. Since
no free DNs-PSS was detected at the ratio 40,13 we may conclude
that all polymer is assembled into the particle, indicating a high
affinity for the polymer. The results obtained at DNs-PSS : CP =
400 support this; although the yield of the smaller particle is lower,
presumably due to a polyelectrolyte effect, no larger particles are
observed. Therefore, in the presence of an excess of polyanion,
only the T = 1 particle is generated. Calculations show that 60
subunits make up the shell of this T = 1 particle, yielding an
icosahedron with 12 pentameric faces (Fig. 5).14
9 J. B. Bancroft, E. Hiebert, M. W. Rees and R. Markham, Virology,
1968, 34, 224–239 and references cited therein. Virus isolation:; J. W.
Davies and B. J. M. Verduin, J. Gen. Virol., 1979, 44, 545–549; protein
isolation:; B. J. M. Verduin, FEBS Lett., 1974, 45, 50–54.
10 K. Matyjaszewski and J. Xia, Chem. Rev., 2001, 101, 2921–2990.
11 G. Woeste, W. H. Meyer and G. Wegner, Makromol. Chem., 1993, 194,
1237–1248.
12 TEM samples of the particles were stained using 5% w/v ammonium
molybdate.
13 This ratio is defined as the amount of polymer repeating unit per
monomeric protein subunit.
In conclusion, we have demonstrated that the self-assembling
properties of CCMV CP lacking part of its N-terminus can be
employed to create a monodisperse 16 nm T = 1 icosahedral
nanoparticle upon encapsulation of a disperse polymer at pH 7.5.
Whether or not non-degraded, or a mixture of partially- and
non-degraded protein exhibits the same behaviour is still under
investigation. In the experiments presented above, no free DNs-
PSS was detected; we may therefore conclude that the equilibrium
of the self-assembly process strongly favours the T = 1 particle.
Since inclusion of this relatively short PSS only yields the T =
1 particles it is feasible that higher molecular weight polymers
14 J. Tang, J. M. Johnson, K. A. Dryden, M. J. Young, A. Zlotnick and
J. E. Johnson, J. Struct. Biol., 2006, 154, 59–67 and references cited.
15 T. Lin, J. Mater. Chem., 2006, 16, 3673–3681.
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