Molecular interactions of DNA with transfectants: a study based on infrared spectroscopy and quantum chemistry as aids to fluorescence spectroscopy and dynamic light scattering analyses

Abstract

Cationic polymers are promising non-viral agents because of their ease of use, inexpensiveness and favorable safety profile. Being inherently cationic, they do spontaneously assemble in water with nucleic acids to give polyplexes. The more popular techniques and methods routinely applied for the characterization of such nanosupensions, unfortunately, do not afford direct observation of anioncation interactions. We thus tackle the issue of probing at the molecular level the interplay occurring between the PO 4 À groups of double stranded DNA and the NH 3 + groups of the simple and representative transfectant poly(L-lysine). By means of IR spectroscopy and Density Functional Theory (DFT) calculations we assigned the red shift of the anti-symmetric PO stretching wavenumber to the intermolecular water-mediated NH 3 +-PO 4 À interaction. These changes observed in moving from ineffective, low nitrogen (N) to DNA phosphate (P) ratios (N/Ps) to more effective conditions are in overall good agreement with changes in polyplex behavior unveiled by means of other analytical techniques such as dynamic light scattering, laser Doppler micro-electrophoresis, and fluorescence spectroscopy, but provide direct insight into the basic intermolecular DNA:transfectant interplay. The application of IR spectroscopic analysis to other non-viral gene delivery vectors may become an appealing approach to disclose molecular details on PO 4 À-cationic polymer interaction, taking into account conformational constraints and steric hindrance effects that the other techniques actually overlook.

Non-Viral in Vitro Gene Delivery: It is Now Time to Set the Bar!

“…Because non-viral gene delivery particles are formed by electrostatic interactions [111,124], they are sensitive to the composition of the medium (i.e., the saline composition, the ionic strength, and the pH) in which the complexation occurs. The most widely used buffers for complexation are 10 mM Hepes [62,96], whether supplemented or not with 5% (w/v) glucose (hereafter referred to as HBG buffer) [125], 150 mM NaCl [126,127], and deionized water (dH 2 O or MilliQ) [128].…”

Comparative evaluation and optimization of off-the-shelf cationic polymers for gene delivery purposes

“…It is well known that polyplex formation is first driven by electrostatic interaction between the cationic groups of the polymer and the anionic phosphates of the DNA, 40 but the final condensation process is entropically driven. 2 Thus, the way of preparing polyplexes and the order of mixing the reagents may affect the formation, the physicochemical properties and the transfection efficiency of the resulting particles.…”

Size matters for in vitro gene delivery: investigating the relationships among complexation protocol, transfection medium, size and sedimentation

“…The formation of polyplexes is first driven by the electrostatic interaction between the positive charges of the cationic polymers and the negative charges of the DNA, and finally by entropy 9 10 . Thus, subtle changes in the way of combining reagents may affect the physicochemical properties and the transfection efficiency of polyplexes.…”