Virus-like particles templated by DNA micelles: A general method for loading virus nanocarriers

Article abstract of DOI:10.1021/ja101444j

DNA amphiphile particles template formation of virus capsids and enable their loading.

Full text of DOI:10.1021/ja101444j

Published on Web 05/19/2010
Virus-like Particles Templated by DNA Micelles: A General Method for
Loading Virus Nanocarriers
Minseok Kwak,† Inge J. Minten,‡ Diana-Milena Anaya,§ Andrew J. Musser,† Melanie Brasch,|
Roeland J. M. Nolte,‡ Klaus Mu¨llen,§ Jeroen J. L. M. Cornelissen,*,‡,| and Andreas Herrmann*,†
Department of Polymer Chemistry, Zernike Institute for AdVanced Materials, UniVersity of Groningen, Nijenborgh 4,
9747 AG Groningen, The Netherlands, Radboud UniVersity Nijmegen, Institute for Molecules and Materials,
Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands, Max Planck Institute for Polymer Research, 55128 Mainz,
Germany, and Laboratory for Biomolecular Nanotechnology, MESA+ Institute for Nanotechnology, UniVersity of Twente,
PO Box 217, 7500 AE Enschede, The Netherlands
Received February 18, 2010; E-mail: j.j.l.m.cornelissen@tnw.utwente.nl; a.herrmann@rug.nl
Scheme 1. DNA Micelle-Templated VC Formationsa
Virus capsids (VCs) or virus-like particles are a relatively new
class of natural biomaterials with great potential for materials
science and nanotechnology. They form precisely defined stable
cage structures, permit coat protein (CP) manipulation through
mutagenesis or chemical modification,1 and can be easily produced.
Such exceptional characteristics make them particularly strong
candidates for applications in biomedicine.2-7 The natural container-
like properties of viruses, as well as their ability to specifically
target individual cells, have been attractive for gene delivery and
are now being harnessed for therapeutic delivery. While some VCs
have been investigated and chemically modified to probe targeting
behavior,8 scant work has been dedicated to loading these nano-
containers.9 An excellent model system in this regard is the Cowpea
a (A) Loading of hydrophobic molecules (green) into the core. (B)
Chlorotic Mottle Virus (CCMV). As with other VCs, CCMV
Equipping moieties (red) attached to complementary DNA by hybridization.
evolved to encapsulate and transport RNA; in its natural state it
Coat proteins encapsulate the micelle by a simple mixing process at neutral
consists of 180 identical CPs, which self-assemble around the central
pH.
RNA into 28 nm icosahedral particles. These can be described
according to the Caspar and Klug T (triangulation) number as T )
Two classes of DNA amphiphiles known to self-assemble into
3 particles.10 It has been shown that such capsids can also be readily
larger aggregates should be distinguished, though representatives
made to self-assemble around large polyanions, resulting in smaller
of both were investigated in this study. The first class consists of
T ) 1 icosahedral particles of 18 nm.11,12 CCMV is unique in that
low molecular weight hydrophobic molecules that are attached to
capsid assembly can be induced in acidic conditions even in the
ODNs.15 Here a lipid-DNA 11mer (UU11) containing two 5-dodec-
absence of nucleic acids, allowing the possibility of loading more
1-ynyluracil nucleobases at the 5′- end was synthesized (Scheme
diverse cargos such as enzymes.13 Nonetheless, the porous walls
S1). The other class of DNA amphiphiles involves linear DNA
of the shell severely complicate the loading and retention of small
block copolymers (DBCs) in which a nucleic acid sequence is
molecules. A still more severe limit is imposed by solvent
covalently connected to a hydrophobic organic polymer via
incompatibilities, which prevent the loading of hydrophobic drugs
complementary end groups.16 This study employed two DBCs
except through covalent modification of the protein or complexation
containing polypropylene oxide blocks of the same molecular
with polyanions.14 The success of engineered virus nanoparticles
weight (MW ) 6800 g/mol) but different lengths of ODNs, namely
as delivery vehicles will hinge in large part on the resolution of
an 11mer (P11) and a 22mer (P22) sequence. See Figure S2 for
these issues and the development of efficient loading strategies for
the DNA sequences and key chemical structures.
small molecules and macromolecular entities.
All three amphiphiles formed micellar structures at room
We report here a strategy for the facile self-assembly and loading
temperature, and in some cases the micelles were additionally
of CCMV capsids using DNA amphiphiles. These structures
loaded with model cargo molecules (SI.5). Particle sizes were
aggregate into micelles with a hydrophobic core and an anionic
characterized by dynamic light scattering (DLS) and AFM, and all
fell in the range 7-11 nm (Figures S4 and S5).15
To assess the ability of these DNA particles to template VC
DNA corona. The negatively charged particles induce capsid
formation, allowing the entrapment of a large number of small
oligonucleotides (ODNs) as a constituent part of the micellar
formation both components were combined through a simple mixing
template. Furthermore, preloading of the micelles with hydrophobic
procedure. In most experiments DNA amphiphiles were mixed with
entities in the core or hydrophilic entities by sequence-specific
CP in a 1:2.3 molar ratio at pH 7.5 and incubated for 30 min at 4
°C. It should be stressed that under these conditions VC formation
can only be attributed to the organizing role of the micelles. The
hybridization enables encapsulation of various small molecules
inside VCs.
resulting materials were isolated by fast protein liquid chromatog-
† University of Groningen.
raphy (FPLC). Transmission electron microscopy (TEM) analysis
revealed successful envelopment of all DNA micelle species by
the CCMV capsid protein. Particles eluted at 1.3-1.4 mL (Figure
‡ Radboud University Nijmegen.
§ Max Planck Institute for Polymer Research.
| University of Twente.
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7834 J. AM. CHEM. SOC. 2010, 132, 7834–7835
10.1021/ja101444j  2010 American Chemical Society

C O M M U N I C A T I O N S
S7A) and exhibited a size of 19.9 ( 3.1 nm (Figure 1A). This size
suggests that the objects have T ) 2 symmetry. Also, a small
proportion of the particles formed a T ) 1 architecture. The TEM
micrographs provide additional evidence of VC loading, since
empty cores would appear dark under the negative staining
conditions (inset Figure 1A).13 Moreover, a fraction that eluted at
1.6 mL could be clearly identified as the remaining UU11 micelles
from its size distribution, 8.1 ( 1.6 nm (Figure 1B), in agreement
with DLS measurements.
virus-like particles that were chemically modified with nucleic acid
sequences at the outside.17 Incorporation of pristine ODNs into VC
was demonstrated for the polyomavirus. However, the loading
procedure was cumbersome because packaging required osmotic
shock treatment as well as an acidic pH.18 The approach presented
herein is much simpler since a high number of ODNs are
preassembled by the attached hydrophobic units, i.e. alkyl chains
or polymers, acting as an efficient soft matter template and avoiding
the need of altering the assembly conditions. Moreover, copackaging
of various small compounds can be achieved by either hybridizing
them onto the micelles or incorporating them into the core. Our
novel loading approach therefore marks a significant step toward
virus-based targeted therapeutics. This is especially true for
hydrophobic drugs, which are difficult to couple chemically to
water-soluble capsid proteins due to solvent incompatibilities. With
the general loading strategy presented here, it is now possible to
fully explore diverse applications, especially the potential of these
nanocarriers as high-impact drug delivery systems.
Acknowledgment. M.K., A.J.M., and A.H. were supported by
the EU (ERC starting grant, ECCell), The Netherlands Organization
for Scientific Research (NWO-Vici), the Nuffic, the DFG, and the
Zernike Institute for Advanced Materials. R.J.M.N. and J.J.L.M.C.
acknowledge the NWO-CW, the Royal Netherlands Academy for
Arts and Sciences (KNAW), and the European Science Foundation
(ESF) for financial support.
Figure 1. TEM micrographs, stained by uranyl acetate, of UU11-VC FPLC
fractions eluted at (A) 1.4 mL, VCs, and (B) 1.6 mL (see Figure S6B),
UU11 micelles. The inset shows empty VCs for comparison. Scale bars
are 40 nm.
After confirming the loading of VCs with DNA amphiphile
aggregates, these scaffolds were further exploited for incorporation
of other moieties. Since hydrophobic compounds are known to
accumulate within the hydrophobic core of DBC amphiphiles, the
fluorescent aromatic compound pyrene was introduced into UU11
aggregates as a model small hydrophobic compound. Templated
assembly of VCs was subsequently carried out as described above.
During the FPLC elution of pyrene-loaded UU11-VC (Figure S7A),
a fraction eluted at ∼1.3 mL showed clear pyrene absorption. The
fluorescence spectrum of the chosen fraction showed sharp and well-
resolved pyrene emission bands demonstrating the presence of the
fluorophore within the UU11 aggregates and thus also inside the
VCs (Figure S7B). Similar measurements were carried out using
micelles loaded with a membrane staining dye, DiI (Figure S6B).
The hydrophobic dye-loaded VCs were further examined by silver-
stained gel electrophoresis (Figure S8) and TEM, which cor-
roborated the analysis above for pristine micelle-loaded VCs.
Finally the challenge of loading small hydrophilic compounds
was addressed. DNA micelles can be equipped by hybridization
with almost any compound if conjugated by means of a cDNA
sequence. Many functionalities are commercially available or can
be easily synthesized. As a proof of concept, UU11 micelles were
labeled with ROX, a hydrophilic fluorescent dye, by hybridization
with a ROX-DNA conjugate. VCs were formed employing the same
general encapsulation procedure. Again FPLC analysis confirmed
successful envelopment of the functionalized micelles (Figure S6A).
With the help of the ROX label and Dylight functionalized CP,
the aggregation number Z of micelles as well as the DNA content
within the VC could be calculated. It turned out that Z amounted
to 25 ( 2, which is in very good agreement with a geometrical
calculation (SI.4). Assuming a T ) 2 configuration, the ODN
content to capsid ratio was found to be 6% by weight.
Supporting Information Available: Experimental details. This
material is available free of charge via the Internet at http://pubs.acs.org.
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Short ODNs have already been combined with virus-like
particles. VC networks were formed by hybridization employing
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