Polymyxin B immobilized on cross-linked cellulose microspheres for endotoxin adsorption

Highlights

• Cross-linked cellulose microspheres were successfully prepared.

• Polymyxin B was immobilized on CL-CMs to synthesize endotoxin adsorbent.

• Adsorption capacity of endotoxin in physiologic saline solution was 3600 EU/g.

• Adsorption efficiency of endotoxin in human plasma was more than 70%.

• Adsorbents showed low nonspecific adsorption of 8.9% for human plasma protein.

Abstract

Cross-linked cellulose microspheres (CL-CMs) were successfully prepared by inverse crosslinking suspension method. NaOH/urea aqueous solution was used as solvent to dissolve cellulose at low temperature. The microspheres presented good spherical shape and monodispersity, which were applied to synthesize endotoxin adsorbent with polymyxin B (PMB) as ligand. The adsorbent showed good adsorption capability on endotoxin in physiologic saline solution and the maximum adsorption capacity was 3605 EU/g (1EU = 100 pg). It was worth noting that more than 70% of endotoxin could be effectively removed from the human plasma with the initial concentration of endotoxin ranged from 1 EU/mL to 5 EU/mL. The dynamic adsorption efficiency of endotoxin was 72.3% at the plasma perfusion rate of 300 mL/h with the endotoxin concentration of 4 EU/mL, while the variation of plasma protein before and after adsorption was only 8.9%. It suggests that the PMB immobilized CL-CMs have great potential application in clinical blood purification.

Introduction

Endotoxin is the component of the outer cell wall of Gram-negative bacteria, which is also called as lipopolysaccharide (LPS). Endotoxin consists of a lipid component (lipid A), a core oligosaccharide and O-specific polysaccharide region (Gorbet and Sefton, 2005, Raetz and Whitfield, 2002). Like other amphipathic molecules, LPS molecules will be inclined to aggregate into larger supra-molecular structures above a particular critical concentration (Erridge, Bennett-Guerrero, & Poxton, 2002). Endotoxin molecules are rather thermostable and their biological activity could be destroyed only after being heated at 160 °C for 2–4 h. Normally, traces of endotoxin play an important role in immunoregulation to enhance the non-specific immunity of the organism. However, when they go right into the bloodstream, endotoxins can cause strong biological reactions (Lieder et al., 2013, Siegemund and Sauer, 2002), such as fever, disseminated intravascular coagulation, hypotension, metabolic disorders and so on (Rietschel et al., 1996). Endotoxin can stimulate mononuclear cells and macrophages to produce cytokines such as interleukin-1, tumor necrosis factor, interferon, macrophage inflammatory protein-1 and leukocyte pyrogen which trigger systemic inflammatory response and lethal shock (Baumgartner and Glauser, 1993, Hanasawa et al., 1997), further leading to multiple organ dysfunction syndromes. The clinical methods for endotoxemia treatment include pharmacotherapy and blood purification therapy. Compared with pharmacotherapy, blood purification is a much more promising way since it does not cause the tissue damage and bacterial death (Bhattacharjya, 2010). Hemoperfusion is one of the most commonly used approaches for blood purification in clinical, where the endotoxins and immune mediators could be removed by selective adsorption when the blood passes through the hemoperfusion apparatus (Takeyama et al., 2012).

The adsorbents used to eliminate endotoxins are expected to bind endotoxin selectively with a low non-specific adsorption. Polymyxin B (PMB) is a polypeptide with 10 molecules of amino acids, which could bind lipid A as the active center of endotoxin not only through ionic bonds between its amino groups and the phosphate groups of lipid A but also hydrophobic association between their hydrophobic groups. However, PMB has neurotoxicity and nephrotoxicity, which limits its application for intravenous administration. Therefore, immobilizing PMB on a carrier to adsorb the endotoxin in blood selectively has become a research hotspot (Suzuki & Shoji, 2010). Novelli et al. used polymyxin B-based hemoperfusion (PMX-DHP) to remove endotoxin in kidney and liver transplantations, the patients presented good graft function and low levels of endotoxin (Novelli et al., 2012). However, the PMB immobilized column with macroporous polystyrene fibers (PMX-F) as carrier is expensive and most of the patients cannot afford the high treatment fee. Therefore, it is important to develop alternative endotoxin adsorbents with both low cost and excellent absorption performance to meet the demand of clinical application.

Cellulose is the most abundant natural polysaccharide and has attracted more and more attention due to its biodegradability, non-toxicity and biocompatibility (Fukuzumi et al., 2009, Klemm et al., 2005). Cellulose gel microspheres possess high surface areas, high mechanical strength, outstanding chemical stability, low non-specific adsorption and active groups (Wang et al., 2014, Yan et al., 2002, Zhang et al., 2012), so it is an ideal carrier material for endotoxin adsorbents. The dissolving of cellulose is a severe problem for its strong inter- and intra-molecular hydrogen bonds and van der Waals forces. What's more, most solvents for cellulose are poisonous (Kruder, 1985, Saalwachier and Burchard, 2001). Recently, Zhang et al. developed novel solvents to dissolve cellulose rapidly at low temperature with aqueous solution containing LiOH or NaOH and urea or thiourea (Cai and Zhang, 2005, Luo and Zhang, 2013, Mao et al., 2006), in which no toxic gas generates during the dissolution process.

In this study, we prepared the cross-linked cellulose microspheres (CL-CMs) from NaOH/urea aqueous solution via inverse crosslinking suspension method. Endotoxin adsorbents were synthesized successfully by immobilizing PMB onto the CL-CMs after activation with 1,4-butanediol diglycidyl ether. The adsorption performance of endotoxin in both physiologic saline and human plasma were studied to evaluate the possibility of CL-CMs for applications in the blood purification field.