Prediction of the appropriate size of drug molecules that could be released by a pulsatile mechanism from pH/thermoresponsive microspheres obtained from preformed polymers

Abstract

Preparation of cross-linked pH/thermoresponsive microspheres from preformed polymers is still lacking in literature since copolymers possessing both temperature- and pH-sensitive units together with a cross-linkable moiety in appropriate ratios are required. Moreover, choosing of the appropriate drugs able to be loaded and then released in a pulsatile manner is randomly performed. Here, we report the synthesis of pH/thermoresponsive cross-linked microspheres based on N-isopropylacrylamide and N-alloc-ethylenediamine. A chromatographic method was developed to predict the appropriate size of drug molecules that could be loaded and then released in a pulsatile manner. Accordingly, it was established that common drugs (salicylic acid, benzoic acid, nicotinic acid, lidocaine and diclofenac), with molecular weights ranging between 100 and 1000 g mol⁻¹, could be loaded and released in a pulsatile manner. Biologic molecules with higher molecular weights (heparin, lysozyme and bovine serum albumin) are completely excluded from the pores of cross-linked pH/thermoresponsive microspheres both below and above the volume phase transition temperature.

Introduction

Stimuli-responsive polymers are a class of materials that undergo a phase transition when small changes in the environmental parameters take place [1], [2], [3]. Among stimuli-responsive polymers, pH- and temperature-responsive polymers are the most used for biomedical applications because they use the change in pH and temperature of the human body as triggering agents in drug delivery [4], [5], [6]. Poly(N-isopropylacrylamide) (poly(NIPAAm)) is the most popular thermoresponsive polymer since it exhibits a sharp phase transition around 32 °C [7], [8]. The temperature at which this transition occurs is called the lower critical solution temperature (LCST). Below the LCST, the polymer chain is hydrated and adopts an extended coil conformation, while above it the polymer is dehydrated and adopts a globular conformation. Correspondingly, the cross-linked hydrogels obtained from these polymers swell under the LCST and shrink above it [9], [10]. This swelling/shrinking process is usually used to control the delivery of drugs in a pulsatile manner [11], [12]. When a pH-sensitive monomer (weak acidic or basic monomer) is copolymerized with N-isopropylacrylamide (NIPAAm), a hydrogel with both pH- and thermosensitive properties is obtained [13]. Moreover, if the pH-sensitive monomer is hydrophilic, the LCST of the copolymer could be increased towards the body temperature [14]. However, conventional thermoresponsive hydrogels are limited for practical applications because of their slow swelling and deswelling rates [15], [16]. Basically, the rates of swelling and deswelling processes of conventional hydrogels depend on the rate of solvent diffusion and on the gel size and porosity [17], [18]. The smaller the size of the hydrogel, the faster the response rate to the input signal. Methods for the preparation of small microspheres with a relative fast swelling/deswelling rate are well known [19], [20].

Most microspheres are prepared from monomers by suspension [11] or precipitate polymerization [21]. The main disadvantage of these methods is that it is more difficult for demonomerization to take place in a tridimensional network than in a solution of linear polymer. Moreover, the removal of monomers is often incomplete.

Thermoresponsive microspheres from preformed polymers can be prepared by dropping a polymer solution into a liquid at a temperature above the LCST [22], [23]. However, these microspheres are not stable or easy to handle, and have a reduced number of biomedical applications. Preparation of microspheres by cross-linking the functional amido groups of poly(N-isopropylacrylamide-co-acrylamide) was pioneered by our research group. However, due to the low reactivity of the amido groups, the necessary time to produce stable microspheres is very long [24]. The loading of these microspheres with biologically active compounds and the release studies were randomly performed [11]. The size of drug molecules is not correlated with the pore size of thermoresponsive microgels below and above the LCST. Therefore, no a priori information is available about the loading and release mechanism, and attempts must be done before.

In this paper, the preparation of stable pH/thermoresponsive microspheres from well-characterized preformed polymers is reported. Poly(N-isopropylacrylamide-co-N-alloc-ethylenediamine) (poly(NIPAAm-co-NAEDA)) was prepared as a new pH/thermoresponsive polymer possessing cross-linkable amino groups and having an LCST tailored towards body temperature. This copolymer was transformed into pH/thermoresponsive stable microspheres by an original approach based on the cross-linking of the amino group of N-alloc-ethylenediamine (NAEDA) with glutaraldehyde (GA) at a temperature slightly below the LCST. The microspheres were characterized by optical and scanning electron microscopy (SEM) in the dried, swollen and shrunken states. Inverse size exclusion chromatography (ISEC) was used to characterize the permeability of microspheres below and above the LCST, and to predict the appropriate size of drug molecules that can be released by a pulsatile mechanism.