Condensing DNA with poly(amido amine) dendrimers of different generations: means of controlling aggregate morphology

Abstract

The morphology of the aggregates formed between DNA and poly(amido amine) (PAMAM) dendrimers depends on the dendrimer generation as previously reported in separate studies at high dendrimer/DNA charge ratios (>1). This has lead to substantial work on dendrimers as possible transfection agents. Inspired by these studies, we here present novel results from a coherent and systematic study using cryo-TEM, dynamic light scattering (DLS) and fluorescence spectroscopy to reveal how the size, composition and morphology of aggregates formed between DNA (4331 base pairs) and PAMAM dendrimers, are affected by dendrimer size and charge at low charge ratios (<1) in dilute solutions. At such conditions the process is cooperative and kinetically controlled and welldefined structured aggregates are formed for lower dendrimer generations. The smaller sized dendrimers (generation 1 and 2), which have a lower total charge per molecule, allow the formation of well-structured rods and toroids. In contrast, globular and less defined aggregates, which are less stable against precipitation, are formed with higher generation dendrimers. We were also able to directly visualise the cooperative nature of the condensation process as cryo-TEM and DLS show that dendrimer/DNA aggregates, containing condensed DNA, coexist with free extended DNA chains. In fact, the apparent hydrodynamic radii of the dendrimer/DNA aggregates, obtained using DLS, are found to be almost constant for charge ratios #1. The fluorescence study shows that the number of dendrimers bound per DNA chain decreases with the dendrimer generation but is independent of the charge ratio.

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

“…On the other hand, very closely placed rate constants of G1 ( k 1 , k 2 and k 3 ) are observed for the charge ratios under study clearly showing the weakest interaction of G1 with pDNA molecules. 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 ).…”

“…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 . The resulting information on dendrimer‐DNA interaction pathways plays an extremely vital role in gene transfection, involving the cargo release kinetics from the vector at the target site . Figure shows kinetic plots of EtBr fluorescence intensity decay observed upon PAMAM dendrimer of generation G1, G2 and G3 binding to pDNA at Z +/‐ 5 as well as 10, in the presence of 10 mM sodium phosphate buffer.…”

Structural variations in hyperbranched polymers prepared via thermal polycondensation of lysine and histidine and their effects on DNA delivery

“…More recent studies have utilised a very wide variety of functionalised cationic lipids (Viricel et al, , Altnoglu et al, , Semple et al, ) and cationic polymers, with increasing efficacy in transfecting target cells with reduced toxicity (Luo et al, , Blum et al, , Lee et al, , Cho et al, ). In this context, polymers based on polyamidoamine, polyethylenimine (PEI), and polylysine (PLL), synthesised into varying architectures from linear to branched and dendritic, have been designed and developed as efficient gene carriers (Khandare et al, , Ainalem et al, , Dufes et al, , Hardy et al, , Bansal et al, , Sun and Davis, ). However, the syntheses of some of these materials are difficult to scale up, rendering the pharmaceutical applications of such materials more difficult.…”

Computer Simulation of DNA Condensation by PAMAM Dendrimer

“…Among them, the ssDNA molecules were located between the PAMAM dendrimers and act as cross‐linkers, as shown in Figure b. The rod‐like DNA/dendrimer aggregates were observed by Ainalem et al at low dendrimer/ssDNA charge ratios in dilute solutions. When the charge ratio reached to = 1.5, large but irregularly shaped aggregates formed (Figure c), and some free dendrimers appeared in the aqueous solution.…”