Chain conformation: A key parameter driving clustering or dispersion in polyelectrolyte – Colloid systems

Abstract

The work presents the characterization of Pluronic F127 micellar solutions in presence of hyaluronic acid in semi-dilute regime. The effects of the nature and salt concentration are investigated by differential scanning calorimetry and small angle neutron scattering. Hyaluronic acid reduces the critical micellar temperature to the same extend as an increase of the ionic strength. Within the investigated HA concentration range, the size and shape of the micelles are not modified by the addition of HA but their dispersion state depends on the salt concentration. By increasing the ionic strength we observe the formation of small micellar clusters which organize into a face-centered cubic liquid crystalline phase at high salt concentration. This behavior is reinforced by increasing the HA concentration or molecular weight. The nature of the salt plays also a role and divalent cations such as Ca²⁺ promote the clustering of micelles and their crystallization. The origin of the aggregative behavior is the change of the HA chain conformation –from stretched to coil- by addition of salt which in turn induces an excluded volume around the micelles and exerts a depletion interaction.

Introduction

In the medical field and especially for cancer treatment, nanocarrier systems with specific targeting are promising tools to reduce the dose and the side effects, to avoid burst release and prolong the circulation into the blood stream. In addition, they must be easily injectable, biocompatible and biodegradable. Among all the potential carriers, Pluronics copolymers are widely investigated. They are composed by two hydrophilic poly(ethylene oxide) (PEO) groups and a central hydrophobic poly(propylene oxide) (PPO) group. Their self-assembling properties (size and shape of the micelles, critical micellar concentration and temperature) can be varied by changing the EO/PO ratio and group lengths. F127 (EO₁₀₀PO₇₀EO₁₀₀) used in this work is involved in a considerable number of patents. The high capability of F127 micelles to load hydrophobic molecules, and more specifically drugs is widely recognized [1].

Hyaluronic acid (HA) is a natural linear semirigid polyelectrolyte with a repeating negatively charged disaccharide unit composed of N-acetyl-glucosamine and glucoronic acid. HA is found in large quantities in animal and human body. It is a common component of synovial fluid and extracellular matrix. However it plays ambiguous roles. Whereas high molecular weight HA (>500 kDa) is anti-angiogenic, anti-inflammatory and immunosuppressive, low molecular weight (10–500 kDa) is highly angiogenic and pro-inflammatory. HA is currently investigated as a target-specific material since many malignant cancer cells overexpress HA receptors and its incorporation in nanocarrier systems can be used as targeting ligand [2].

Considering the relevant properties of each of these macromolecules, several studies report on the potential applications of the mixed systems for tissue engineering and drug delivery. The presence of HA improves the mechanical and mucoadhesive properties and permits a sustained release of drugs [3], [4], [5], [6], [7], [8]. These studies are carried out mostly in the range of concentration where the Pluronic micelles form a gel i.e. at concentrations above 15 vol%, in the hexagonal phase. A detailed structural characterization of these complex systems is often lacking but dynamic light scattering characterization shows the formation of structures of few hundreds of nanometers [6], [9], i.e. more than ten times the micellar size. Before any therapeutic application, the knowledge of the structure appears mandatory, especially if aggregation occurs and might be an issue for injection and circulation into the blood vessels.

From a fundamental point of view, the phase behavior of small colloids and large polymers has attracted much interest in the past years due to its importance in many domains (from nanocomposites to care products) and more specifically for applications in protein crystallization giving to such polymer-colloid systems the name of “protein limit”. The counterpart -large colloids and short polymers- is called “colloidal limit”. In the latter case, the natural parameter to introduce is the Q parameter defined as R_G/R, the ratio between the radius of gyration of the polymer and the radius of the colloid [10], [11]. The colloidal limit is simpler and can be understood by the fact that the polymers are excluded from the space between two adjacent colloids, and exert an unbalanced osmotic pressure which induces an attraction between the particles. In other words, the depletion of the polymers around particles causes entropy loss of polymer when the particles are homogeneously dispersed. The aggregation of the particles increases the accessible volume for the polymers and leads to an entropy gain. In this case, depletion interaction are pairwise additive. The radius of gyration fixes the range of depletion and the polymer concentration sets the strength. The model first proposed by Asakura-Oosawa [10], [11] and further developed by Vrij [12] gives accurate results for polymer in θ solvent and Q < 1. In the protein limit and in a semi-dilute polymer solution, the correlations between the segments of the polymers induce correlations between the positions of the particles which behave as if they were attracted to each other [13]. The theoretical treatment and the phase diagram forecast are more difficult since many-body colloidal interactions are involved and there is in addition the necessity to take into account the polymer-polymer interactions and the chain conformation. The first scaling theories were developed by Sear et al. [14], [15], [16]. The main hypotheses are the following: 1- the polymer is considered to be in good solvent conditions and does not adsorb on the particles; 2- all the interactions considered are excluded volume interactions, i.e two polymer segments or two particles or a polymer segment and a particle cannot occupy the same volume. The demixing occurs if the entropy of mixing decreases due to a loss of entropy of translation for the particles or a loss of entropy of conformation for the polymer. For a dilute polymer solution, the chains do not overlap and the system is miscible. When the polymer concentration reaches c* (semi-dilute regime), the chains start to overlap and form a network with a characteristic mesh size or correlation length ξ [13] in which the particles can enter. In consequence, in the protein limit and for polymer in semi-dilute regime, the particle-polymer interaction depends on the parameter Q defined here as the ratio D/ ξ (where D is the particle diameter) and scales with the polymer volume fraction. As the concentration of polymer increases, ξ decreases and the space available to the particle decreases until $\xi ~D$. In that limit, the free energy to insert a particle and push away the polymer becomes large and drives the phase separation [16]. For $D<\xi$ and low particle concentration, the position of the particles in the polymer solution are correlated over the polymer correlation length as if they were subject to an additional attractive potential. When the particle concentration increases, the correlation between the polymer segments are in turn modified by the particle inducing a drop of the correlation length [17], [18], [19]. Further theoretical developments were done within the PRISM (Polymer Reference Interaction Site Model) theory [20], [21], which allowed direct determination of thermodynamic and structural properties of the colloid/polymer mixtures. More recently, full–monomer simulations were able to describe the phase boundary for Q ≥ 1 [22]. Based on the free-volume theory of Lekkerkerker [23] and Bolhuis [22], Fleer and Tuinier [24], [25] have derived a general law that allows to predict the binodals and generate a universal scaling law, regardless the polymer and the colloid size.

Most of the experimental studies on depletion concerns nonionic polymers with charged colloids but few is known about the structure and interaction of polyelectrolytes with nonionic colloids. For potential applications as drug delivery systems, it is also mandatory to control the aggregation state and the stability, two factors that may affect the therapeutic efficiency. With this perspective, we are interested in the mixed system composed of Pluronic F127 micelles and HA. Since F127 and HA do not exhibit specific adsorption nor electrostatic interaction, a depletion phenomenon should be observed depending on the polyelectrolyte concentration or the ionic strength that screens the inter- and intra-chain electrostatic repulsions. Two HA, with molecular masses of 12 and 300 kDa are used. We work in the Pluronic micellar phase (3 vol%) and different HA volume fractions above 1%, in the semi-dilute regime. The effects of the nature of the salt and its concentration on the formation, shape and organization of the micelles are probed by differential scanning calorimetry (DSC) and small angle neutron scattering (SANS).