Yi-Chun Liu 1,2 , Chun-Jen Su 1 , Hsin-Lung Chen 1* , Hsien-Kuang Lin 2, Wen-Lian Liu 2 and U-Ser Jeng 3
1 Department of Chemical Engineering, National Tsing Hua University, Taiwan
2 Materials Research Laboratories, Industrial Technology Research Institute, Taiwan
3 National Synchrotron Radiation Research Center, Hsin-Chu, Taiwan
ABSTRACT
The self-assembly mechanism and the resultant structures of the complexes of DNA with surface protonated poly(amidoamine) (PAMAM) dendrimers of generation two (G2) and four (G4) have been studied as a complex[1-2]. However, in a recent study of the phase behavior of DNA complexes with poly(propylene imine)
(PPI) dendrimers of intermediate sizes (i.e.,G4 and G5)
[3], Heather et al. revealed the structural transitions function of the molar ratio of dendrimer surface charge to
DNA base pair (x). For both dendrimer systems soluble undercharged complexes were formed at x < 1.5 without obvious sign of DNA ordering in the complexes. between columnar mesophases with in-plane square and hexagonal symmetries, which greatly modified the commonly assumed “beads-on-string” model.
In spite of the previous efforts, further studies are necessary to resolve the detailed mechanism associated
Significant aggregation of the complexes resulting in precipitation occurred at x > 1.5, yielding condensed mesomorphic phases in which the DNA was orientationally and/or positionally ordered. The condensed phase in G2 dendrimer complexes was characterized by the nematic ordering of DNA. DNA/G4 dendrimer complexes exhibited a square columnar structure at x > 3 in addition to the condensed nematic with the structural formation of dendrimer/DNA complexes. In this study, the effects of several key parameters such as the dendrimer-to-DNA molar ratio and the generation of the dendrimer on the binding mechanism and the self-assembled structure of the complexes are examined. In contrast to the study by
Heather et al., the present study centers on the PAMAM dendrimers of both small (G2) and intermediate (G4) phase appeared at smaller x. Complexation with DNA deformed the dendrimer molecules due to mismatch between the surface curvature of DNA and that of dendrimer molecules under the condition of maximizing their electrostatic interactions. Upon incorporating metallic nanoparticles into the core region of dendrimer, the subsequent complexation with DNA may generate ordered nanoparticle arrays.
INTRODUCTION
Starburst dendrimers constitute a special class of macromolecule characterized by their compact and highly symmetric molecular structure composed layers of monomer units irradiating from a central core.
Polyamidoamine (PAMAM) starburst dendrimers, for instance, are obtained by covalently attaching amidoamine units to an amino or ethylenediamine core.
Each complete grafting cycle is called a “generation”.
Dendrimers are biocompatible and can be used in the biochemical fields as for example vechicles of biological materials and probes for oligonucleotide arrays. In particular, positively charged dendrimers have been complexed with DNA to act as gene vectors for gene therapy.
Cationic dendrimers are effectively macroions; thus their complexation with DNA may induce “DNA condensation” through which the molecularly dissolved worm-like DNA chains in aqueous solution aggregate into compact ordered condensates. Various models have been proposed for the self-assembled structures of dendrimer/DNA complexes. It was used to believe that the DNA chains tended to coil around the dendrimer molecules in the complexes, resembling the supramolecular configuration of DNA/histone sizes to reveal the size effect on the self-assembly behavior of the complexes. Furthermore, we will demonstrate how the complexation may be used to direct the assembly of gold nanoparticles into ordered arrays.
EXPERIMENTAL
Linear DNA type XIV from herring testes sodium salt was purchased from Sigma and used without further purification. PAMAM dendrimers of generation 2 (20 wt% in methanol) and generation 4 (10 wt% in methanol) were obtained from Aldrich. To complex with the polyanionic DNA, the surface amino groups of PAMAM dendrimers were first protonated by adding prescribed amount of 0.2 M HCl. Because the basicity of the surface primary amines is significantly larger than the interior tertiary amines, it is reasonable to assume that the surface amino groups of the dendrimer were fully protonated first.
The protonated dendrimer aqueous solution was mixed with the aqueous solution containing prescribed amount of DNA to obtain the complex. The occurrence of complexation was visually identifiable by precipitation for most compositions. The complex composition, x, is expressed by the molar ratio of dendrimer surface charge to base pairs in DNA.
RESULTS AND DISCUSSION
Self-Assembled Structures. For both dendrimer systems soluble undercharged complexes are formed at x < 1.5 and the system is said to form a “non-condensed phase”
Significant aggregation of the complexes resulting in visually observable precipitation occurred at x > 1.5, and the system is said to form “condensed phase”. The SAXS profiles of the complexes in the non-condensed (x < 1.5) and condensed (x > 1.5) phases are displayed in Figure 1.
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Let us consider the scattering patterns of G4 system first.
The SAXS profiles associated with the non-condensed phase is characterized by a monotonically decayed curve, indicating the lack of coherent inter-chain correlation of
DNA. As x is increased to 1.5 and 2 (i.e., as the system enters the condensed phase regime), a rather broad
DNA-DNA correlation peak at q * =1.5 nm -1 becomes visible. The location of this scattering peak corresponds to the interhelical distance of DNA, d
DNA
= 2

/q*

4.2 nm. It is noted that these compositions display optically birefringerent patterns characteristic to liquid crystalline phases; therefore the corresponding condensed phase is attributed to a nematic phase ( cf.
Figure 2(a)) in light of the lack of higher-order diffraction peaks in the SAXS profiles. Higher-order diffraction peaks appear at x > 3, signaling the formation of long-range ordered morphology. The relative positions of the scattering peaks closely follow the ratio of 1:2 1/2 :4 1/2 , corresponding to the lattice scattering from a square columnar phase ( cf.
Figure 2(b))with a lattice constant of 4.2 nm.
In the condensed phase regime, G2/DNA complexes exhibit a single broad scattering peak (corresponding to d
DNA
= 3.7 nm) associated with a nematic phase irrespective of x. The surface groups of the smaller G2 dendrimers are indeed more mobile than those of G4 dendrimers due to the more opened structure. The larger flexibility in structure allows for greater electrostatic interaction with DNA which is unfavorable in terms of dynamics for the interior reorganization of DNA to attain a more ordered structure. Therefore, a compact and tight structure such as square columnar phase was not formed under the present experimental time scale.
It is noted that the hexagonal symmetry reported by
Heather et al. for polypropylene (imine) (PPI) G4 dendrimer/DNA complex [3] was not observed here. The surface positive charges of PAMAM G4 dendrimer and
PPI G4 dendrimer are 64e and 32e, respectively. The stronger dendrimer-dendrimer repulsion due to highly charged surface groups of PAMAM dendrimer is likely to stabilize the square columnar phase over the hexagonal phase 19 . d
DNA in G4 complexes is about 4.2 nm. The size of a
G4 dendrimer molecule in the complex is then 2.2 nm after deducting the diameter of DNA (2.0nm) from the observed interhelical distance. However, the diameter of a G4 dendrimer molecule is 4.5 nm. This means that the dendrimer molecules are somehow deformed to a spheroid upon binding to DNA. Similar degree of deformation is observed for the G2 molecules in the complex. The deformation may be driven by the enhancement of the effectiveness of charge matching with the DNA phosphate groups in that DNA is a planar molecule which is not geometrically favorable for the adhesion of the sphere-like dendrimer molecules with large curvature. The dendrimer molecules hence deform and become flatter to enhance the electrostatic interaction with DNA.
Nanoparticle Assembly Directed by the Complexation.
In addition to its potential use as gene carrier,
DNA/dendrimer complex is also an attractive material for nanostructure construction. Let us consider a case where the complexation directs the assembly of gold
10
5
10
3
10
1
10
-1 nanoparticles embedded within the dendrimer. Gold nanoparticles with the size of 1 ~ 5 nm were first synthesized using PAMAM dendrimer as the template by reducing HAuCl
4
.salt incorporating in the core of dendrimers. The surface of the dendrimer molecules enclosing the nanoparticles was subsequently protonated and finally mixed with DNA to generate the complex forming condensed phase. It is hoped in this case that the ordering of DNA induced by the complexation can direct the assembly/organization of the dendrimer-embedded nanoparticles. Figure 3 displays the morphology of the resultant complex viewed under AFM. It can be seen that nanoparticle arrays are indeed formed. It is interesting to note that the inter-particle distance along the long axis of the array is about 3.4 nm, which corresponds well to the pitch of a B-form DNA duplex.
REFERENCES
[1] Ottaviani, M. F. et al. Macromolecules 1999, 32,
2275.
[2] Chen, W. et al. Langmuir 2000 , 16 , 15.
[3] Heather M. et al. Phys. Rev. Lett.
2003 , 91 ,7,075501.
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Figure 1. SAXS profiles of (a) G4/DNA and (b) G2/DNA complexes
3.0
Figure 2. Schematic illustrations of (a) nematic and (b) square columnar phases in dendrimer/DNA complexes
Figure 3. Arrays of dendrimer-embedded gold nanoparticles in the dendrimer/DNA complex.
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