Polyelectrolyte complex formation and stability when mixing polyanions and polycations in salted media: A model study related to the case of body fluids

Abstract

Controlled drug delivery and gene transfection involve contact of artificial polyelectrolytic systems that can interact dramatically with biopolymers and cells when they are introduced in blood. Given the complexity of body aqueous media in terms of physical chemistry, a model approach was selected in attempt to understand the behavior of artificial polyelectrolytes introduced in body fluids. Selection in terms of molecular weight was highlighted in a previous paper. In the present study the formation and the stability of fractions obtained when a polycation is added to a polyanion according to a titrating process mimicking injection into blood was considered for different polycation/polyanion couples. Poly(amino serinate) and poly(l-lysine) were used as polybases, and poly(acrylic acid), poly(l-lysine citramide) and poly(l-lysine citramide imide) as polyacids. Four fractions corresponding to different positive/negative charge ratios were formed for each couple. At low polyion concentration (13 mg/L) and given salt concentration, the stability of the complex fractions depended on molecular weight and charge density of the polyions. The NaCl concentration required to destabilize the different interpolyelectrolyte complexes was found to decrease from the first fraction to the fourth one. Upon decreasing the salt concentration, macroscopic flocculation occurred in the case of PLL/PAA complex fractions only. For the other couples, dynamic light scattering showed that several hundreds nanometer sized particles were formed that were stable in a broad range of NaCl concentration, including the physiological 0.15 ionic strength. At higher polyion concentrations, stable solid precipitate was formed regardless of the system. The absence of flocculation in the case of highly diluted poly(l-lysine citramide) and poly(l-lysine citramide imide) polyanions in salted media is assigned to the presence of non-ionic hydroxyl and amide polar groups along the complexed chains. Data show that introducing non-ionic functions along the polyelectrolyte chains is a good means to keep interpolyelectrolyte complexes dispersed in salted media, a conclusion of interest in the field of condensation of genes by polycations.

Introduction

It is well known that polyions of opposite charges interact electrostatically with each other to form polyelectrolyte complexes or IPECs (Fuoss and Sadek, 1949). The formation and the properties of IPECs depend on various factors including nature and position of the ionic groups, charge density and concentration, proportion of opposite charges, molecular weight of the macromolecules and physicochemical environment (Tsuchida et al., 1974, Nakajima, 1980, Tsuchida, 1980, Tsuchida, 1994, Bekturov and Bimendina, 1981, Tsuchida and Abe, 1982, Philipp et al., 1989, Kötz et al., 1996).

IPECs play an important role in nature where many charged systems, namely proteins, polysaccharides and cells are in contact in body fluids and tissues. Nowadays, drug delivery and gene therapy involve positively and negatively charged macromolecules in many instances (Vert, 1982, Vert, 1986, Behr, 1994, Kabanov, 1994, Kabanov and Kabanov, 1995, Luo and Saltzman, 2000). Artificial polyelectrolytic carriers of bioactive compounds, such as drugs and genes, can interact with the various charged species present in body fluids and may lead to dramatic phenomena like cell aggregation or hemolysis (Katchalsky et al., 1959, Moreau et al., 2000, Moreau et al., 2002, Mao et al., 2004, Fischer et al., 2004). Therefore, it is of great importance to understand the behavior of artificial and natural charged macromolecules when they become in presence after introduction in an animal body. Several variables affect the formation mechanisms and the stability of IPECs in water, namely the strength of the polyelectrolytes in terms of acid or base, the molecular weight of the polyions and the pH and the ionic strength of the medium (Kabanov et al., 1985, Dautzenberg et al., 1994, Dautzenberg and Karibyants, 1999, Dragan and Cristea, 2002, Leclercq et al., 2003, Zintchenko et al., 2003, Seyrek et al., 2003, Becheran-Maron et al., 2004, Dragan and Schwarz, 2004, Dufresne and Leroux, 2004). The knowledge of the effects induced by salt on IPECs is critical in the search of carrier systems for drugs or genes. Only a few studies regarding selectivity phenomena related to the polymolecularity of artificial polymers were reported in literature, probably because of the complexity of performing precise analysis of IPECs (Kabanov et al., 1980, Kabanov et al., 1985, Izumrudov et al., 1988a, Izumrudov et al., 1988b, Izumrudov et al., 1995, Izumrudov et al., 1996, Jo et al., 1997, Harada and Kataoka, 1999, Ruponen et al., 2001, Leclercq et al., 2003, Boustta et al., 2004, Wahlund et al., 2004). Recently, some of us demonstrated by an original method based on affinity chromatography that fractionation occurred when a polycation taken as a standard is added to a solution of a polydisperse polyanion. The high molecular weight polyanionic macromolecules precipitate first, whereas the shorter ones remain in the solution phase (Boustta et al., 2004).

In the present article, we wish to complement our investigation by the characterization of IPECs formed during the blending of various polyanions, namely poly(l-lysine citramide) or PLCA, poly(l-lysine citramide imide) or PLCAI and poly(acrylate) or PAA and two polycations, namely poly(l-lysine) or PLL and poly(amino serinate) or PSA by dynamic light scattering (DLS). PLCA and PLCAI polymers are artificial multifunctional polyelectrolytes composed of building blocks based on alternating l-lysine and citric acid moieties and thus bearing hydroxyl groups along the chains (Scheme 1) (Boustta et al., 1991, Huguet et al., 1991, Couffin-Hoarau et al., 2001). PSA is an artificial polyester-type polyelectrolyte based on d,l-serine moieties (Scheme 1) (Rossignol et al., 1999). First, the formation and the behavior of the IPECs were studied as a function of the nature of the polyions and the ionic strength of the medium. Second, four IPEC fractions were prepared from the different polyions by a titration-type protocol. This was a simple means to mimic the situation when IPECs are formed during injections of polyelectrolytic systems in body fluids. DLS was used to assess the size and stability of the complexes in the presence of salt at various concentrations with a special attention to 0.15 M that is known to correspond to the ionic strength in blood.