J. Phys. Chem. B 2010, 114, 2667–2675
2667
Ability to Adapt: Different Generations of PAMAM Dendrimers Show Different Behaviors in Binding siRNA Giovanni M. Pavan,*,† Lorenzo Albertazzi,‡,§ and Andrea Danani† UniVersity for Applied Sciences of Southern Switzerland (SUPSI) - Institute of Computer Integrated Manufacturing for Sustainable InnoVation (ICIMSI), Centro Galleria 2, Manno, CH-6928, Switzerland, NEST, Scuola Normale Superiore and CNR-INFM, 56127 Pisa, Italy, and IIT@NEST, Center for Nanotechnology InnoVation, 56127 Pisa, Italy ReceiVed: January 11, 2010
This paper reports a molecular dynamic study to explore the diverse behavior of different generations of poly(amidoamine) (PAMAM) dendrimers in binding siRNA. Our models show good accordance with experimental measurements. Simulations demonstrate that the molecular flexibility of PAMAMs plays a crucial role in the binding event, which is controlled by the modulation between enthalpy and entropy of binding. Importantly, the ability of dendrimers to adapt to siRNA is strongly dependent on the generation and on the pH due to backfolding. While G4 demonstrates good adaptability to siRNA, G6 behaves like a rigid sphere with a consistent loss in the binding affinity. G5 shows a hybrid behavior, maintaining rigid and flexible aspects, with a strong dependence of its properties on the pH. To define the “best binder”, the mere energetic definition of binding affinity appears to be no longer effective and a novel concept of “efficiency” should be considered, being the balance between enthalpy and entropy of binding indivisible from the structural flexibility. With this aim, we propose an original criterion to define and rank the ability of these molecules to adapt their structure to bind a charged target. Introduction The design of binding agents devoted to create high-affinity complexes with nucleic acids is a key point in the development of nanocarriers for gene therapy and, in particular, the study of their multivalent recognition ability to strengthen binding is extremely important to generate systems with potential biomedical applications.1 Recently, dendritic structures has been proposed as ideal candidates to deliver and release genetic material inside cells.2,3 These molecules offer relevant advantages in binding nucleic acids owing to their multivalent structure.4 A dendrimer is a highly branched macromolecule with a spherical shape that grows in generations.5 Its ordered structure is characterized by strong symmetry and periodicity and the surface can be functionalized in many ways to modulate the physicochemical properties of the final construct.6,7 Poly(amidoamine) (PAMAM) dendrimers are the most widely studied dendritic structures.8,9 Pioneering studies of Tomalia and Szoka on nucleic acids binding dendrimers evidenced the ability of these polycationic structures to create a strong and stable binding with nucleic acids and to achieve gene delivery into cells.3,9-11 The reason of such high affinity of PAMAM dendrimers toward DNA and siRNA is settled in their multivalent polycationic surface that is able of multiple strong ionic interactions with the charged phosphate groups of nucleic acids.12 Previous studies on multivalency demonstrated that the binding action of more ligands at the same time is a cooperative phenomenon. In fact, once the first ligand has bound to the target, the binding of a second ligand requires a lower entropic cost resulting in a stronger interaction.13 Since the number of * To whom correspondence should be addressed. E-mail: giovanni.pavan@ supsi.ch. † SUPSI - ICIMSI. ‡ Scuola Normale Superiore and CNR-INFM. § Center for Nanotechnology Innovation.
charged surface groups grows exponentially with the dendritic generation, the global attraction with nucleic acids is higher for larger dendrimers. Together with the binding affinity, gene delivery profiles also usually improve at higher dendritic generation, for example, PAMAM dendrimers exhibit optimal gene delivery at about the fifth generation of growth (G5).14 However, previous characterization on PAMAM dendrimers evidenced that higher generation shows more consistent backfolding and steric crowding within the structure.15 Therefore, the question of how many of these charged surface groups are actively participating and how many are simple “spectators” in binding is a key point in the study of the multivalent behavior of these molecules. In a recent work, we evidenced that the second generation of dendrons functionalized with spermine can use few of its surface groups to preserve high DNA binding affinity by screening the disturbing action of ions in high salt concentrated solution.16 On the other hand, the first generation system had not enough active sites to maintain high binding affinity under the same conditions. This demonstrates that the multivalent binding action of multiple surface groups cannot be explained as a simple attraction (it is a balance between the amount of charge and ability to use these charges efficiently) but it is related to the molecular flexibility. There is a limit in the advantage that is possible to obtain simply adding charged groups to the structure that depends on the surface density and makes the difference in treating dendrimers as flexible molecules rather then as rigid spheres. Previous experimental and computational studies on PAMAMs were focused mainly on the structural characterization of these dendrimers.17-19 The first computational approaches to understand the binding between PAMAM dendrimers and nucleic acids demonstrated that at high generations of growth single-stranded DNA is able to roll up on dendrimer surface.20 However, the understanding of the mechanism of binding
10.1021/jp100271w 2010 American Chemical Society Published on Web 02/10/2010
2668
J. Phys. Chem. B, Vol. 114, No. 8, 2010
Pavan et al.
Figure 1. Molecular models (and sequence) of GL3 siRNA (a) and of PAMAM dendrimer (G4+, b) used for simulations. Within G4+, the central (CEN) residue is colored in red, the repetitive (REP) branch units in white and the surface groups (END) are represented in green.
remained poor. Moreover, there was disagreement between modeling and SAXS and SANS experimental measurements on the determination of the size of these PAMAM dendrimers. In fact, while scattering analyses report a substantial independency of the radius of gyration (Rg) on pH changes,21 previous atomistic22 and coarse-grained23 simulations show a consistent increase of Rg going from high (∼10) to low (
