Interaction of bovine serum albumin with self-assembled nanoparticles of 6-O-cholesterol modified chitosan

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Abstract

In order to understand the nanomedicine and nanotoxicological effects of self-assembled nanoparticles of 6-O-cholesterol modified chitosan (O-CHCS NPs) as a carrier for drug delivery systems, the interaction between O-CHCS NPs and bovine serum albumin (BSA) was studied by spectroscopy and calorimetric methods. The morphology of the complex between O-CHCS NPs and BSA observed by transmission electron microscope (TEM) was almost spherical shape. The size and the zeta potential of the complex increased with the concentration of O-CHCS NPs increasing. The fluorescence spectroscopy indicated that the micro-environment around the tryptophan (Trp) residues in BSA had slight change due to only partially exposure of the Trp residues to water in the interaction process. Compared with free BSA, the addition of O-CHCS NPs led to the decrease of α-helical content of BSA and the increase of β-strand content. Isothermal titration calorimeter (ITC) results showed that the binding reaction between O-CHCS NPs and BSA was exothermic and enthalpically driven. Therefore, it could be concluded that hydrogen bonding, hydrophobic and electrostatic interactions played a key role in the complex formation, and the formation mechanism was proposed accordingly. In addition, cytotoxicity assay implied that O-CHCS NPs were non-cytotoxic and biocompatible up to 200 μg mL−1. These data demonstrated the potential application of O-CHCS NPs for drug delivery.

Highlights

► The interaction between O-CHCS nanoparticles and BSA was studied by spectroscopy and calorimetric methods. ► Hydrogen bonding, hydrophobic and electrostatic interactions existed in the interaction process. ► The complex formation mechanism was proposed. ► O-CHCS nanoparticles were biocompatible.

Introduction

The development of appropriate vehicles for delivery of proteins, peptides, oligonucleotides genes and anticancer drugs is of growing interest in the field of biomedical application, because they can reduce unwanted toxic side effects and improve the therapeutic effect [1], [2]. Among various vehicles, amphiphilic polymeric nanoparticles have been shown to possess significant potential as a carrier for drug delivery, especially based on natural polysaccharides [3], [4], [5], [6], [7] due to their outstanding physicochemical and biological properties including non-toxicity, biocompatibility, biodegradability and self-assembly property in aqueous solution. Nevertheless, nanoparticles that enter the body may interact with proteins such as human serum albumin (HSA), as proteins can bind a wide variety of ligands such as metal ions, surfactants, drugs and nanoparticles in living systems [8], [9], [10]. The interaction may have severe effects not only on the protein function and conformation [11], [12] but also on the physicochemical properties of nanoparticles including the size, zeta potential, surface structure, shape and aggregation [13], [14], [15]. However, the study on the potential biological risks from nanoparticle therapeutic application has seldom been done so far [16], [17]. Therefore, it is necessary to investigate the interaction between proteins and nanoparticles, such as amphiphilic polymeric nanoparticles, to discover the role of nanoparticle drug carriers on biological and toxic response.

Previously, Akiyoshi et al. [18] studied the thermodynamics of complexation between cholesterol-bearing pullulan (CHP) nanoparticles and bovine serum albumin (BSA) by isothermal titration calorimetry (ITC). Moreover, our group reported that the degrees of substitution (DS) of hydrophobic groups significantly affected the interaction between BSA and self-assembled nanoparticles of cholesterol-grafted O-carboxymethyl chitosan (CCMC) modified through amino groups [19]. Nevertheless, the modification of chitosan through amino groups could change the fundamental skeleton of chitosan, and the modified chitosan lost the original physicochemical and biochemical activities [20]. It was suggested that chitosan modification through hydroxyl groups might have an advantage because there might be less influence on the fundamental skeleton [21] and it preserved free amino groups of chitosan which were responsible for many of chitosan advanced functions, including biological activity and cationic polymer properties [22]. Additionally, different substitution site of hydrophobic groups might have influence on the interaction between nanoparticles and proteins, because intermolecular interactions including hydrogen bonding, van der Waal forces, electrostatic and hydrophobic interactions could be altered by different functional groups and substitution positions. Based on the above consideration, we synthesized 6-O-cholesterol modified chitosan (O-CHCS) conjugates with succinyl linkages (Fig. 1) in order to retain the fundamental skeleton and free amino groups of chitosan, and prepared O-CHCS self-assembled nanoparticles (O-CHCS NPs) by the dialysis method [23] without the need of high temperature, organic solvents, surfactants or other special experimental technology, which was in favor of avoiding the degradation of proteins or hydrophobic drugs in the process of nanoparticle fabrication. The as-prepared O-CHCS NPs were expected as an ideal carrier in drug delivery systems.

In this study, BSA was chosen as a model protein because of its medical important and its tertiary structures which were similar to human serum albumin (HSA) in 76% [24]. The interaction between O-CHCS NPs and BSA was investigated systemically by spectroscopy and thermodynamic methods, and the formation mechanism of the complex between O-CHCS NPs and BSA was proposed according to intermolecular interaction. Moreover, the cytotoxicity of O-CHCS NPs against HeLa cells was assayed to further demonstrate the biocompatible property of O-CHCS NPs.

Section snippets

Materials

6-O-Cholesterol modified chitosan (O-CHCS, chitosan molecular weight was 105 Da and DS of cholesterol groups was 4.0%) was synthesized according to a previously reported procedure [23]. Bovine serum albumin (BSA) was purchased from Sigma Chemicals (St. Louis, MO). 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium sodium salt (WST-1) was obtained from Beyotime Institute of Biotechnology (Jiangsu, PR China). Dulbecco's modified Eagle's medium (DMEM) was purchased from

Morphology and zeta potential

In this study, O-CHCS self-assembled nanoparticles (O-CHCS NPs) were prepared by dialysis. Fig. 2A showed the typical TEM image of O-CHCS NPs in almost spherical shape with good structure integrity and the mean diameter of O-CHCS NPs measured by DLLS (Table 1) was 187.4 ± 6.17 nm. These results implied that O-CHCS could spontaneously form self-aggregate with a hydrophilic outer-shell of chitosan backbones and a hydrophobic core of cholesterol moieties in aqueous media. The zeta potential of O-CHCS

Conclusions

The interaction between O-CHCS NPs and BSA was investigated by spectroscopy and calorimetric methods. The results indicated that the conformation of BSA was not significantly changed in the interaction process, and the binding force between O-CHCS NPs and BSA was mainly driven by hydrogen bonding, hydrophobic and electrostatic interactions. TEM showed that the morphology of the complex between O-CHCS NPs and BSA was still nearly spherical in shape. In addition, the interaction between O-CHCS

Acknowledgements

This research was supported by Major Research Plan of National Natural Science Foundation of China (90923042), Doctoral Fund of Ministry of Education of China (20101106110042), and National Key Scientific Projects of China (2006CB933300).

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