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Artificial Virus
DOI: 10.1002/anie.200800266
Filamentous Artificial Virus from a Self-Assembled DiscreteNanoribbon**
Yong-beom Lim, Eunji Lee, You-Rim Yoon, Myeong Sup Lee, and Myongsoo Lee*
The creation of virus-like nanomaterials (artificial viruses)has been the subject of intensive research in the field of gene/drug delivery because of their huge therapeutic potential.
[1]
Some progress has been made in the field; however,compared to the natural viruses, which are evolution-tailoredexperts in gene delivery, the synthetic system is still far behindthe natural system. One of the critical reasons for thisshortcoming is that the size and shape of the artificial viruses,among the most important determinants of their efficiency,are very difficult to control.As the polyanionic nature of nucleic acids (DNA andRNA) prevents them from crossing the same chargedcytoplasmic membrane barrier, a vector system is necessaryto neutralize their charge and to install other functions, suchas cell binding, endosome escape, and nucleus localization.Although there are serious safety concerns, such as immuno-genicity and carcinogenesis, in the use of viral vectors, theyare still far more widely used for gene therapy than nonviralvectors (artificial viruses) because of their higher efficiency.
[1a]
The basic principle of artificial virus formation is acondensation reaction which is induced by the attractionbetween oppositely charged molecules (for example, betweencationic polymer and DNA).
However, this polyion cou-pling generally leads to the formation of huge nanoaggregatesthat are highly heterogeneous in size and shape, and isuncontrollable in most cases.
[3]
In regard to the size issue, acertain optimal size exists for the nanoparticulate deliverysystems to work best. The shape of the nanoparticle can alsobe a crucial factor. For example, sometimes the cylindrical(that is, filamentous) nanostructure has certain advantagesover the spherical one in that it persists longer in vivo,
[4]
whichmight explain why many filamentous viruses exist in nature.Therefore, it is imperative to find a general strategy to controlthe size and shape of artificial viruses.Recent advances in supramolecular chemistry have madeit possible, by rational design of building blocks, to controlsupramolecular architectures from spherical micelles, cylin-drical micelles, vesicles, and toroids to nanotubes.
[5]
Inspiredby this elaboration, we envisioned that the control of the sizeand shape of artificial viruses would be possible if thepreorganized supramolecular architectures used were robustpolycationic scaffolds that remain unchanged after theformation of an interpolyelectrolyte complex (IPC) withnegatively charged nucleic acids. Herein, we report a poten-tially generalizable strategy to create an artificial virus thatmemorizes the size and shape of its precursor. By using apreorganized supramolecular nanostructure as a template, weshow that well-defined, discrete artificial viruses can beelaborated after IPC formation between the nanostructureand nucleic acids. This controlled feature and appropriatesurface functionalization with multivalent carbohydrateligands make the artificial virus highly efficient in theintracellular delivery of genes and drugs.With the aim of constructing filament-shaped, discreteartificial viruses, a
b
-sheet peptide-based supramolecularbuilding block (Glu-KW) was designed (Figure 1a). To ourknowledge the filament-shaped artificial virus is unprece-dented. It has been shown that the combination of hydro-phobic and electrostatic interactions produced by the alter-nating placement of hydrophobic and charged amino acids in
b
-sheet peptides promotes
b
-sheet interactionand subsequentself-assembly into bilayered filamentous nanostructures(
b
ribbon).
[6]
Coupling of hydrophilic segments, such aspolyethylene glycol, hydrophilic peptides, or carbohydrates,to
b
-sheet peptides has been reported to stabilize
b
-ribbonnanostructures by suppressing lateral aggregate forma-tion.
The Glu-KW structure is characterized by a
b
-sheet-forming self-assembly segment, two linker segments, a nucleicacid-binding cationic segment, and a carbohydrate ligandsegment. The
b
-sheet peptide segment consists of trypto-phan–lysine–tryptophan–aspartic acid repeats, the amino acidconfiguration of which promotes
b
-sheet formation. Thelinker segments are designed to be flexible and nonionic byusing glycine and serine residues. The eight lysine residues areplaced between two linker segments to shield the cationicsegment, upon self-assembly, from the
b
-ribbon surface.
d-
Glucose is positioned at the outermost part of Glu-KW torender the
b
-ribbon surfacecharge-neutralandtoincreasethechances of
b
-ribbon binding to the cell surface throughmultivalent interactions
with cell-surface glucose trans-porters (GLUTs). GLUTs are present in nearly all mamma-lian cells and overexpressed in most cancer cells.
[8]
To address the question of whether Glu-KW forms
b
-sheet-mediated nanostructures, the self-assembly of Glu-KWwas investigated by circular dichroism (CD) and transmission
[*] Dr. Y.-b. Lim, E. Lee, Y.-R. Yoon, Prof. M. LeeCenter for Supramolecular Nano-Assembly andDepartment of ChemistryYonsei University, Seoul 120-749 (Korea)Fax: (
+
82)2-393-6096E-mail: mslee@yonsei.ac.krHomepage: http://csna.yonsei.ac.krDr. M. S. LeeDepartment of Biochemistry, Yonsei University (Korea)[**] We gratefully acknowledge the National Creative Research InitiativeProgram of the Korean Ministry of Science and Technology forfinancial support of this work.Supporting information for this article is available on the WWWunder http://www.angewandte.org or from the author.
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electron microscopy (TEM). The studies were performed inphosphate-buffered saline (PBS), a physiological buffer. Thenegative minimum of molar ellipticity at 213 nm in the CDspectrum indicates
b
-sheet interaction (Figure 1d). Indeed,the
b
-sheet interaction promoted the formation of discrete
b
ribbons, as revealed by TEM investigation (Figure 1c). Thetypical length of the
b
ribbon was relatively short at about70 nm, which is likely to be advantageous for intracellulardelivery applications.We next asked whether the
b
ribbon can make IPCs withnegatively charged small interfering RNA (siRNA) throughthe positively charged regions of the lysine residues. ThesiRNA is double-stranded RNA that is 21–23 nucleotides inlength and induces sequence-specific post-transcriptionalgene silencing called RNA interference (RNAi).
RNAi isa powerful gene silencing process that holds great promise inthe field of gene therapy.
The siRNA gradually lost itselectrophoretic mobility as the relative amount of the
b
ribbon increased, thus indicating that
b
ribbon/siRNAcomplexes were formed (Figure 2a). The charge neutraliza-tion of the siRNA was achieved at a charge ratio (
+
/
À
) of around 2. Remarkably, the TEM image of the
b
ribbon/siRNA complexes showed that the
b
ribbon maintained itsdiscrete and short nanoribbon morphology even after siRNAcomplexation (Figure 2b). Atomic force microscopy (AFM)investigation revealed that the height of the
b
ribbon grewfrom 0.67 to 0.85 nm after siRNA addition, which indicatesthat
b
ribbon/siRNA complexes were formed (Figure 2c,d).
[9]
Upon mixing the Glu-KW
b
ribbon with siRNA, thepositive CD band of the siRNA at 261 nm increased andunderwent a long-wavelength shift of 19 nm (see the Sup-porting Information). These CD changes are characteristic of double-stranded RNA in a condensed state,
and indicatethat the siRNA binds directly to the cationic segment of theGlu-KW
b
ribbon. In addition, a CD experiment with double-stranded DNA (dsDNA) of the same size as the siRNAshowed that the intensity of the negative CD band of thedsDNA at 250 nm increased and the positive CD band at279 nm underwent a long-wavelength shift (see the Support-ing Information). These CD changes represent the character-istic transition of the DNA structure from the B type to the
Y
-DNA type.
The formation of
Y
-DNA is an indication of dehydration and neutralization of DNA phosphate groups bypolycations. The evidence clearly indicates that the nucleic
Figure 1.
a) Structure of Glu-KW. A
b
-sheet peptide segment, nonionicsegments (linkers and
d
-glucose), and a cationic segment are shownin blue, green, and yellow, respectively. Glu:
d
-glucose. For structuresof all the building blocks (Glu-KW, NH
2
-KW, FAM-KW, Man-RKW, andGlu-RKW; FAM
=
carboxyfluorescein, Man
=
mannose), see the Sup-porting Information. b) Molecular model of the artificial virus incorpo-rating small interfering RNAs (siRNAs; blue, double-helix shape) andhydrophobic guest molecules (red). c) TEM image of a Glu-KW
b
ribbon. Scale bar: 100 nm. d) CD spectrum of Glu-KW (15
m
m
inPBS).
Figure 2.
Formation of the artificial virus (
b
ribbon/siRNA complex).a) Change in electrophoretic mobility (4% agarose gel) of greenfluorescent protein (GFP) siRNA after the addition of increasingamounts of Glu-KW
b
ribbon. The numbers indicate the charge ratio(
+
/
À
). b) TEM image of Glu-KW
b
ribbon/GFP siRNA complexes;
+
/
À
: 2. Scale bar: 50 nm. c,d) AFM images of Glu-KW
b
ribbons (c)and Glu-KW
b
ribbon/GFP siRNA complexes (d);
+
/
À
: 2.
[9]
Diagramsbeneath the images are height profiles along the yellow lines.
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acids (siRNA and dsDNA) interact directly and strongly withthe cationic segment of the Glu-KW
b
ribbon.The hydrodynamic radius (
R
H
) of the
b
ribbon remainedunchanged after complex formation. In stark contrast, the
R
H
of a
b
ribbon self-assembled from a building block devoid of glucose (NH
2
-KW) increased abruptly following siRNAcomplexation (see the Supporting Information). Inter-ribbon cross-linking induced by the addition of negativelycharged siRNA should be responsible for this phenomenon,as the surface of the NH
2
-KW
b
ribbon is densely coated withpositively charged primary amine groups.All these results indicate, as the molecular model inFigure 1b suggests, that the capacity of positively chargedregions created by
b
-ribbon formation is large enough toincorporate relatively small interfering RNA inside, and thatthe glucose-covered charge-neutral surface is crucial inpreventing uncontrollable aggregation of the elementary
b
ribbons. The other building blocks (Man-RKW and Glu-RKW) designed through the same concept as the Glu-KWshowed a similar siRNA complexation behavior, thus indicat-ing that the strategy is potentially generalizable (see theSupporting Information).To address the potential of the Glu-KW artificial virus asan intracellular siRNA delivery carrier, an RNAi experimentwith GFP was conducted on HeLa cells, a human cervicalcancer cell line. As shown in Figure 3a, GFP knockdownexperiments performed in the absence of serum showed thatthe Glu-KW
b
ribbon was even better than Lipofect-amine 2000 (LF2000), a commercial reagent known to haveone of the highest siRNA transfection efficiencies. Thispromising result led us to investigate the gene knockdownefficiency in the presence of serum, a condition representingan in vivo physiological milieu. The results showed that theefficiency of GFP knockdown by the Glu-KW
b
ribbon iscomparable to that of LF2000 (Figure 3b). LF2000 is knownto have high siRNA transfection efficiency in the presence of serum as well. In contrast, the NH
2
-KW
b
ribbon was almostunable to transfect siRNA. Hence, the discrete nanostruc-tures encapsulating siRNAwithin the noncharged surface arecrucial for the high transfection efficiency. Several importantfactors are likely to be synergistically involved in the highsiRNA transfection efficiency of the Glu-KW artificial virus,such as controlled formation of artificial viruses, minimalinteraction with serum proteins by the charge-neutral surface,and enhancement of the cell interaction by multivalentcoating of carbohydrate ligands.As the interfacial hydrophobic space formed within thebilayered
b
ribbon is a suitable place to encapsulate hydro-phobic guest molecules,
we next addressed the question of whether the
b
ribbon can deliver siRNA while encapsulatingsuch guest molecules. For this we first encapsulated ahydrophobic guest (nile red) within a Glu-WK
b
ribbon andthen siRNA was complexed with the Glu-WK
b
ribbon/nilered binary complex. The ternary complex was still able toknock down GFP expression, albeit the efficiency was slightlydecreased compared to that of a Glu-KW
b
ribbon withoutnile red (Figure 3b). Nile red delivered by the ternarycomplex was localized not only in the cytoplasmic compart-ment but also in the nucleus (Figure 3c). The nucleus is thesite of action for many drugs and most anticancer drugs.Therefore, the Glu-KW artificial virus has the potential tobecome an efficient nanomaterial for delivering a hydro-phobic drug and gene simultaneously into the cell nucleus aswell as the cytoplasm.To gain further insight into the intracellular distributionand delivery mechanism, Glu-KW was co-assembled
with afluorescently labeled building block (FAM-KW) to label the
b
ribbon. The image in Figure 3d shows the intracellularpattern of
b
-ribbon distribution (green), which suggests anendocytic entry pathway
[14]
. In addition, co-localization(yellow) of the
b
ribbon and LysoTracker (red) furtherdemonstrates an endocytotic uptake mechanism. LysoTrackeris an acidotropic reagent for labeling and tracing acidicorganelles, such as late endosomes and lysosomes, in live cells.In summary, we have demonstrated that directed assem-bly of an artificial virus is possible by using rationallydesigned, preorganized
b
ribbons. A unique feature of ournanostructure is its capability to encapsulate siRNA withinthe noncharged carbohydrate surfaces while preserving itsdiscrete nanostructure. Considering the variety of supra-molecular nanostructures that are currently or will beavailable, this type of approach provides a general means toconstruct various repertoires of controllable artificial viruses.It is anticipated that other functional properties of naturalviruses could be further finely installed in this artificial virus,
Figure 3.
Intracellular deliveries of siRNA and hydrophobic guestmolecules. a) Knockdown of GFP expression in HeLa cells. Thenumbers in parentheses indicate the amounts of GFP siRNA used.The experiment was performed in the absence of serum (mean
Æ
standard deviation (SD),
n
=
3). b) Knockdown experiment performedwith 40 pmol GFP siRNA in the presence of 10% serum (mean
Æ
SD,
n
=
3). c) Intracellular distribution of nile red (red) delivered by theternary complex of Glu-KW, nile red, and GFP siRNA. The nucleus wasstained with 4
’
-6-diamidino-2-phenylindole (blue). Scale bar: 50
m
m.d) Intracellular distribution studies by confocal laser scanning micros-copy. The Glu-KW/FAM-KW
b
ribbon and LysoTracker Red DND-99 areshown in green and red, respectively. Glu-KW and FAM-KW were co-assembled at 100:1 molar ratio. Scale bar: 50
m
m.
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