Biodegradable and biocompatible polyampholyte microgels derived from chitosan, carboxymethyl cellulose and modified methyl cellulose

Abstract

Two biocompatible and biodegradable polyampholyte microgels, namely chitosan–carboxymethyl cellulose (CS–CMC) and chitosan–modified methyl cellulose (CS–ModMC) were synthesized by an inverse microemulsion technique. The CS–CMC microgel system was pH-responsive while the CS–ModMC system possessed both pH and thermo-responsive properties. For CS–CMC system, the number of –OCH₂COOH and –NH₂ groups was determined to be 1.5 and 1.1 meq/g of microgel, respectively. In the pH range of 4–9, the zeta potential values varied from +10 to −40 mV, while the hydrodynamic radius varied from 160 nm in the swollen state (acidic and basic pH) to 110 nm in the “collapse” state (neutral pH). Furthermore, TEM micrographs confirmed the swelling/deswelling behaviour of CS–CMC microgel particles at acidic, neutral and basic conditions. For CS–ModMC system, the number of –OCH₂COOH and –NH₂ groups was determined to be 0.8 and 0.6 meq/g microgel, respectively. In the pH range of 4–9, the surface charge on the microgels varied from +25 to −60 mV and the hydrodynamic radii were 190 nm at low pH, 80 nm at neutral pH, to 120 nm at a high pH. In vitro drug release studies confirmed that CS–CMC microgels could encapsulate and release a model drug, thus they could potentially be used as biocompatible and biodegradable drug carriers.

Introduction

Polyampholytes are defined as polymers capable of possessing both positive and negative charges on their backbones. The properties of polyampholytes are dependent on intra-chain electrostatic interactions since both negative and positive charges are present on the same backbone (Neyret & Vincent, 1997). Polyampholytes differ from polyelectrolytes in the sense that polyelectrolytes may possess either positive or negative charges on their backbones, however polyampholytes possess both charges on the same backbone. Microgels on the other hand are crosslinked polymer particles with sizes ranging from 100 nm to 1 μm, and they swell in a good solvent (Das & Kumacheva, 2006). Microgels possess functional properties, and depending on the constituent polymers, they exhibit different stimuli-responsive behaviour (swelling/de-swelling behaviour) in response to pH, temperature, ionic strength, solvent and external magnetic field (Tan & Tam, 2008). By combining the properties of ‘polyampholyte’ and ‘microgel’ we obtain a new class of microgel called ‘polyampholyte microgel’. This microgel possesses positive and negative charges, arising from the pH dependent amines and carboxylic acids. At low pH, the amino groups become protonated and acquire positive charges while at high pH, carboxylic acid groups become deprotonated and acquire negative charges.

The primary reason for synthesizing polyampholyte microgels is that their pH responsive behaviour will be attractive for drug and protein delivery applications. Ho, Tan, Tan, and Tam (2008) synthesized polyampholyte microgels using poly(methacrylic acid) (PMAA) and poly(2-(dimethylamino)ethyl methacrylate) (PDMA). Inverse microemulsion polymerization technique was used to polymerize MAA and DMA in the presence of a cross-linker (allyl methacrylate) to produce an amphoteric microgel system. The microgels exhibited swelling at low and high pH while they deswelled at neutral pH. At low and high pH, the microgel particles acquire positive and negative charges, respectively and the particles begin to swell due to the osmotic pressure from counter ions and repulsion from like charges within the microgel particles. Conversely, as the solution approaches a neutral pH, equal number of positive and negative charges on the microgel particles results in a smaller hydrodynamic radius as the counterions leave the microgel particles causing them to deswell. Due to the overall charge neutralization on the surface of the microgel, the particles tend to flocculate at the isoelectric point (IEP). In order to prevent flocculation at IEP, a steric stabilizer, such as poly (ethylene glycol) methacrylate (PEGMA) was grafted on the microgels (Ho et al., 2008, Tan et al., 2007).

Recently, many different polyampholyte microgel systems have been examined (Christodoulakis and Vamvakaki, 2010, Schachschal et al., 2010, Wang et al., 2010, Xu et al., 2010). Due to their potential applications and stimuli responsive characteristics, we have synthesized polyampholyte microgels from biopolymers instead of synthetic polymers. In our approach, we utilize water-soluble polymers dispersed in a continuous organic phase together with a water-soluble crosslinker, such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), to produce crosslinked microgels. This synthetic technique is akin to inverse microemulsion polymerization (IMEP). Simple EDC carbodiimide chemistry was used to crosslink amino and carboxylic acid groups through amide linkages (Nakajima & Ikada, 1995). Inverse microemulsions are thermodynamically stable water-in-oil (W/O) emulsions formed by adding large amounts of surfactants (Oh, Drumright, Siegwart, & Matyjaszewski, 2008). Inverse microemulsion technique is especially useful for water-soluble polymers that tend to polymerize in the aqueous phase. Water droplets in a W/O inverse microemulsion act as nanoreactors, thereby allowing encapsulation and crosslinking of water-soluble polymeric chains in the water nano-droplets.

Non-ionic surfactants are required to produce inverse microemulsions because of their unique phase-inversion characteristic. Non-ionic surfactants transform an oil-in-water (O/W) emulsion at room temperature to water-in-oil (W/O) emulsion at higher temperatures. The temperature, at which phase-inversion occurs for the transformation of an O/W to W/O emulsion is called the phase inversion temperature (PIT). The PIT is a strong function of the hydrophile–lipophile balance (HLB) of surfactants, which corresponds to the ratio of hydrophilic (water-loving) to lipophilic (oil-loving) segments on a surfactant. Mixing two non-ionic surfactants with different HLB values produces a desired HLB value to yield the desired PIT (Lehnert, Tarabishi, & Leuenberger, 1994). Thus, by mixing appropriate amounts of two non-ionic surfactants, a HLB value that gives rise to a desired phase inversion temperature can be achieved.

We have synthesized polyampholyte microgels composed of biodegradable polymers, namely chitosan, carboxymethyl cellulose and methyl cellulose that are derived from natural sources, such as chitin and cellulose. Chitosan (CS) is obtained by the deacetylation of chitin (Rinaudo, 2008), carboxymethyl cellulose (CMC) from carboxymethylation of cellulose (Heinze & Koschella, 2005) and methyl cellulose (MC) from methylation of alkali cellulose (Mansour, Nagaty, & Elzawawy, 1994). Two different polyampholytic systems were explored: chitosan–carboxymethyl cellulose (CS–CMC) and chitosan–modified methyl cellulose (CS–ModMC). In the first system, chitosan and carboxymethyl cellulose were chosen since they would impart amino and carboxylic acid functionalities to the microgels. As a further extension of the CS–CMC system, we have also synthesized chitosan–modified methyl cellulose system where the methyl cellulose was modified by carboxymethylation (Heinze & Koschella, 2005) to incorporate carboxylic acid functionality to the microgels. Methyl cellulose contains methoxide groups, and the hydrophobic interaction between methoxide groups induces chain association at the lower critical solution temperature (LCST) (Chevillard & Axelos, 1997). Thus, by combining the pH responsive characteristics of chitosan and thermo-responsive behaviour of methyl cellulose, a bi-responsive system was produced. The advantages of using these naturally derived polyampholyte microgels are:

• The microgels are biodegradable and biocompatible (Chen and Fan, 2008, Rinaudo, 2008, Yoon et al., 2006)

• They are non toxic and non allergenic (Samir, Alloin, & Dufresne, 2005)

• They are readily available and cheap as they are derived from renewable resources, which can be recycled (Samir, Alloin, & Dufresne, 2005)

• Using polymers as starting material instead of synthetic monomers ensure that toxic initiators are absent in the products, making them suitable for biomedical and pharmacological applications (Agnihotri et al., 2004, Kamel et al., 2008)