Competitive Reactions in Solutions of Poly-l-histidine, Calf Thymus DNA, and Synthetic Polyanions:  Determining the Binding Constants of Polyelectrolytes

Abstract

The physicochemical characteristics of a nonviral gene delivery system will govern its functional bioactivity; however, empiricism dominates the literature in this field, and a significant deficiency of quantitative investigation and evaluation of nonviral gene delivery vehicles remains. Herein, we derive a physical model and experimental method to quantitatively determine the binding constants between a model polycationic nonviral gene delivery vehicle poly-L-histidine (PLH) and calf thymus DNA. The approach has utility to a variety of systems and is not limited to the described polymer model. The interaction of PLH with DNA was monitored by fluorescence quenching of an ethidium bromide probe in the pH range 4 to 8. The interaction increased with pH decrease with the most pronounced change between pH 6 and 7. The obtained pH-dependence of fraction of salt bonds formed between PLH and DNA was used to estimate pK(a) of PLH in the presence of DNA, which equaled 6.24. The interaction of PLH with DNA in the presence of added synthetic polyanions was studied by the same approach and found to be controlled by pH, nature of the charge groups of the polyanion, and its degree of polymerization. In the mixture with sodium poly(styrenesulfonate) the interaction was negligible in the whole studied pH range, whereas in the mixtures with sodium poly(acrylate) (PA) or sodium poly(methacrylate), DNA was able to compete effectively for the binding with PLH. For PA samples with degree of polymerization higher than degree of polymerization of PLH, DP(PA) > DP(PLH), the fraction of polycation bound to DNA was constant regardless of DP(PA.) In contrast, at DP(PA) < DP(PLH), a pronounced increase in the bound fraction was observed. It substantiates the notion that the binding energy of two polymers is mainly controlled by the DP of the shorter component of polyelectrolyte complex. The data on PLH distribution between DNA and added polyanion with different values of DP were treated according to the developed procedure to yield the effective binding constants of PLH with DNA and polyanion-competitor, calculated both per mole of interacting units K(1) and mole of interacting chains K(n). In all cases, K(1) had similar numerical values reflecting common type of interaction stabilizing the complexes, i.e., electrostatics. Slight variation of K(1) yielded in drastic changes in K(n) and alteration of dominance of PLH interaction with DNA or synthetic polyanion. The results of the study can have a high impact in deriving the correlation between the binding constant of a polycation to DNA and its ability to serve as gene delivery vehicle.

Recombinant polymer‐protein fusion: a promising approach towards efficient and targeted gene delivery

“…Considering the full condensation of 1 mole pEGFP with 100 mole (KH) 6 -FGF2 as determined by gel retardation assay, every 1.6 negative charges (N) was neutralized by 1 positive charge (P). Condensation of every 1.6 negative charges with 1 positive charge is perhaps due to the active contribution of histidine residues in DNA condensation via hydrogenbond formation [36,37]. Considering the total number of histidine and lysine residues in DNA condensation calculations, the N/P ratio was determined to be 1 : 1.…”

Effect of ionic strength solution on the stability of chitosan–DNA nanoparticles

“…This can be attributed to unprotonated, neutral chitosan units abstracting protons from the buffer in order to interact with the anionic phosphate groups of the DNA, resulting in proton exchange between the buffer and chitosan [25]. The interaction between DNA and chitosan is exothermic in sodium phosphate buffer [26] and the polyanionic nature of DNA increases the ionisation of chitosan by reducing its surface potential and thereby increasing the polycation pKa [27].…”

The Effect and Role of Carbon Atoms in Poly(β-amino ester)s for DNA Binding and Gene Delivery

“…One challenge in evaluating and optimizing polymer structure is that synthetic polymers can be polydisperse, with variable extents of reaction and molecular weight heterogeneity. 18-20 Isolating precise polymer structures and uniform molecular weight are key to being able to evaluate polymer structure.…”