Ternary interpolyelectrolyte complexes insulin-poly(methylaminophosphazene)-dextran sulfate for oral delivery of insulin.

Ternary interpolyelectrolyte complexes of insulin with biodegradable synthetic cationic polymer, poly(methylaminophosphazene) hydrochloride (PMAP), and dextran sulfate (DS) were investigated by means of turbidimetry, dynamic light scattering, phase analysis, and high-sensitivity differential scanning calorimetry. Formation of ternary insoluble stoichiometric Insulin-PMAP-DS complexes was detected under conditions imitating the human gastric environment (pH 2, 0.15 M NaCl). A complete immobilization of insulin in the complexes was observed in a wide range of the reaction mixture compositions. The ternary complexes were shown to dissolve and dissociate under conditions imitating the human intestinal environment (pH 8.3, 0.15 M NaCl). The products of the complex dissociation were free insulin and soluble binary Insulin-PMAP complexes. The conformational stability of insulin in the soluble complexes of various compositions was investigated by high-sensitivity differential scanning calorimetry. The dependence of the excess denaturation free energy of insulin in these complexes on the PMAP content was obtained. The binding constants of the folded and unfolded forms of insulin to the PMAP polycation were estimated. Proteolysis of insulin involved in the insoluble ternary complexes by pepsin was investigated under physiological conditions. It was found that the complexes ensure an almost 100% protection of insulin against proteolytic degradation. The obtained results provide a perspective basis for development of oral insulin preparations.

Polyplexes of poly(methylaminophosphazene): energetics of DNA melting.

The interaction of DNA with a synthetic biocompatible and biodegradable cationic polymer, poly(methylaminophosphazene) hydrochloride (PMAP·HCl), was investigated by high-sensitivity differential scanning calorimetry under conditions of strong and weak electrostatic interactions of the macroions. Thermodynamic parameters of the DNA double-helix melting were determined as a function of pH and the PMAP·HCl/DNA weight ratio. PMAP·HCL was shown to reveal two functions with respect to DNA: the polyelectrolyte function and the donor-acceptor one. The first function stabilizes the helical conformation of DNA, and the second one destabilizes it. The stabilizing effect of PMAP·HCl is of entropic origin, related to a displacement of mobile counterions from the DNA's nearest surroundings by the poly(methylaminophosphazene) charged groups. The donor-acceptor function of poly(methylaminophosphazene) dominates when its electrostatic interaction with DNA is either saturated (in the complex coacervate phase at high poly(methylaminophosphazene) concentrations) or completely suppressed (in a salt medium when the polycation carries a small charge). Under these conditions, poly(methylaminophosphazene) destabilizes DNA. It preferentially binds to the DNA coil form likely via the formation of multiple labile hydrogen bonds with the donor-acceptor groups of DNA.

Conformational energetics of interpolyelectrolyte complexation between ι-carrageenan and poly(methylaminophosphazene) measured by high-sensitivity differential scanning calorimetry.

The interaction of poly(methylaminophosphazene) hydrochloride (PMAP·HCl) of varying degrees of ionization (f) with the potassium salt of ι-carrageenan was studied by high-sensitivity differential scanning calorimetry at a KCl concentration of 0.15 M, which is included for the purpose of stabilizing the helix conformation of the polysaccharide up to 55 °C. The conditions of strong (pH 3.8, I = 0.15), moderate (pH 7.4, I = 0.15), and weak (pH 7.4, I = 0.25) electrostatic interactions of the polyelectrolytes were considered. The thermodynamic parameters of the helix-coil transition of ι-carrageenan were determined as a function of the polycation/polyanion ratio. We show that the interpolyelectrolyte reaction between PMAP·HCl and ι-carrageenan results in a complete unfolding of the polysaccharide helix under conditions of strong electrostatic interaction and increases its stability under conditions of medium and weak electrostatic interactions. The formation of stoichiometric PMAP-carrageenan interpolyelectrolyte complexes proceeded via a cooperative mechanism at pH 3.8 (f = 0.5) and pH 7.4 (f = 0.2) at an ionic strength of 0.15. In contrast, the complexation at pH 7.4 and an ionic strength of 0.25 could be considered to be a consecutive competitive binding of charged units of poly(methylaminophosphazene) to the oppositely charged polysaccharide matrix in the helix or coil conformation. Binding constants of the polycation to the helix and coil forms of ι-carrageenan were estimated. They revealed a preferential binding of the polycation to the helix form of the polysaccharide.