Stopped-Flow Kinetic Studies of Poly(amidoamine) Dendrimer–Calf Thymus DNA To Form Dendriplexes

Abstract

Poly(amidoamine) (PAMAM) dendrimers are known to be highly efficient nonviral carriers in gene delivery. Dendrimer-mediated transfection is known to be a function of the dendrimer to DNA charge ratio as well as the size of the dendrimer. In the present study, the binding kinetics of four PAMAM dendrimers (G1, G2, G3, and G4) with calf thymus DNA (CT-DNA) has been studied using stopped-flow fluorescence spectroscopy. The effect of dendrimer-to-DNA charge ratio and dendrimer generation on the binding kinetics was investigated. In most cases, the results of dendrimer-CT-DNA binding can be explained by a two-step reaction mechanism: a rapid electrostatic binding between the dendrimer and DNA, followed by a conformational change of the dendrimer-DNA complex that ultimately leads to DNA condensation. It was observed that the charge ratio on the dendrimer and the DNA phosphate groups, as well as the dendrimer generation (size), has a marked effect on the kinetics of binding between the DNA and the dendrimers. The rate constant (k'1) of the first step was much higher compared to that of the second step (k'2), and both were found to increase with an increase in dendrimer concentration. Among the four generations of dendrimers, G4 exhibited significantly faster binding kinetics compared to the three smaller generation dendrimers.

Role of Unmodified Low Generation - PAMAM Dendrimers in Efficient Non-Toxic Gene Transfection

“…The observed insignificancy of G1 rate constants is probably due to the consequence of its smaller size leading to larger surface area and therefore results a lower charge density that enabled the formation of well‐structured dendriplexes . The above described dendrimer‐pDNA binding kinetic pathways is in good agreement with the earlier findings on CT‐DNA binding with amino acid based surfactants as well as PAMAM dendrimer (G1, G2, G3 and G4) and further supports the findings on EtBr and gel condensation assays (Figure ).…”

“…The relative rate constants k 1 , k 2 and k 3 correspond to the binding process of the dendrimer to pDNA to form random pDNA coils followed by DNA compaction and further rearrangement respectively. In general, the reported EtBr displacement mechanism from pDNA by the dendrimer is very similar to earlier studies and the value of relative rate constants are found to vary with dendrimer to pDNA charge ratio as well as the dendrimer generation. Each rate constant shown in Table I is an average of three independent kinetic experiments.…”

“…Here again, most classic works where computational/experimental techniques have been used to focus on higher generation dendrimers. Even though, few recent publications focus on lower generation dendrimers, the existing literature have mostly used huge CT‐DNA molecules and the corresponding rate constants cannot be correlated with our present work where we have used much smaller plasmid DNA. Hence, it becomes important to draw DNA‐dendrimer binding information from the stop‐flow kinetics, which is known for understanding fast reactions, where rapid mixing of reactant solutions is necessary .…”

“…Even though, the condensation of pDNA by the dendrimers of first three generations are demonstrated successfully by EtBr as well as gel condensation assays (Figure (a) and (b)), investigation on pDNA‐dendrimer binding kinetics is also equally essential to understand the pDNA compaction mechanism . Herein, the essentiality of the binding kinetics arises mainly due to our aim in releasing the therapeutic pDNA from the lower generation based dendriplex (G1, G2 and G3) into the cytoplasm of the target cell.…”

“…From Table I, it is evident that for dendrimers of three generations under study, all the three relative rate constants are well separated mostly, by at least an order of magnitude, and, in general, increases with increasing charge ratio for G2 and G3. Based on the previous report, the first step (eq. (4)) is due to the electrostatic binding of the dendrimer on the pDNA molecule, which is a diffusion‐driven process and also faster with increase in the amount of dendrimer concentration.…”