Elsevier

Toxicology in Vitro

Volume 29, Issue 3, April 2015, Pages 638-646
Toxicology in Vitro

Biocompatibility of a self-assembled glycol chitosan nanogel

https://doi.org/10.1016/j.tiv.2014.11.004Get rights and content

Highlights

  • Thorough biocompatibility study of a particular nanogel based on glycol chitosan.

  • Absence of nanogel cytotoxicity confirmed through a set of viability assays.

  • Nanogel safe for systemic application: stealthy properties and hemocompatibility.

Abstract

The research of chitosan-based nanogel for biomedical applications has grown exponentially in the last years; however, its biocompatibility is still insufficiently reported. Hence, the present work provides a thorough study of the biocompatibility of a glycol chitosan (GC) nanogel. The obtained results showed that GC nanogel induced slight decrease on metabolic activity of RAW, 3T3 and HMEC cell cultures, although no effect on cell membrane integrity was verified. The nanogel does not promote cell death by apoptosis and/or necrosis, exception made for the HMEC cell line challenged with the higher GC nanogel concentration. Cell cycle arrest on G1 phase was observed only in the case of RAW cells. Remarkably, the nanogel is poorly internalized by bone marrow derived macrophages and does not trigger the activation of the complement system. GC nanogel blood compatibility was confirmed through haemolysis and whole blood clotting time assays. Overall, the results demonstrated the safety of the use of the GC nanogel as drug delivery system.

Introduction

Nanoparticles have been largely researched as drug nanocarriers, yet their interaction with cells and extracellular environment is still poorly explored. The evaluation of the potential effects of drug delivery devices on the biological systems is indeed a crucial requirement in the development of nanomedicines: cytotoxicity, haematocompatibility (haemolysis and complement activation), inflammatory response; biodegradability and potential cytotoxicity of the degradation products, cellular uptake and intracellular fate, in vivo biodistribution studies, all assist in allowing a better definition of the biological properties of the novel polymers and their polymeric nanoparticles. (Gaspar and Duncan, 2009, Naahidi et al., 2013, Rodrigues et al., 2012) The so called “biocompatibility” is largely dependent on the physical and chemical properties of the nanoparticles (size, shape and surface characteristics), as well as on the used raw material (Liu et al., 2013, Naahidi et al., 2013).

The use of polymeric nanoparticles based on chitosan has been extensively reported in biomedical applications, due to its interesting characteristics. Non-toxicity, biocompatibility, biodegradability, antibacterial activity, mucoadhesiveness and permeation enhancing properties are among the features that have been reported, the cationic character being responsible for some of them (Bernkop-Schnurch and Dunnhaupt, 2012, Croisier and Jérôme, 2013). Although often claimed as biocompatible, more comprehensive studies are required for a proper understanding of the biological effects of this polymer and of its nanoformulations.

As reported previously, a polymeric nanoparticle made of glycol chitosan (GC), here designated GC nanogel, has been synthesized in our laboratory by chemical grafting hydrophobic chains on the GC backbone, yielding an amphiphilic polymer capable of self-assembling in aqueous environment (Pereira et al., 2013). The present work focuses on the detailed evaluation of the biocompatibility of this nanogel. For this purpose, in vitro cell toxicity of the GC nanogel was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release assays to gauge the nanogel cytotoxicity. Induction of apoptosis and/or cell cycle arresting was tested through flow cytometry. The complement activation was semi-quantified by western blot, analysing the degradation of the C3 factor. Interaction with murine macrophages was observed through confocal microscopy. Finally, the nanogel haemocompatibility was evaluated through haemolysis and whole blood clotting time assays.

Section snippets

Materials

Glycol chitosan (GC, G7753), mercapto hexadecanoic acid (MHDA), N-hydroxysulfosuccinimide (NHS) and 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), NADH, pyruvate, haemoglobin from bovine blood and Drabkin’s reagent were acquired from Sigma–Aldrich. 5/6-Carboxyfluorescein succinimidyl ester was purchased from Thermo Scientific. Cell Culture reagents and culture medium were purchased from Biochrom.

Nanogel self-assembling

GC nanogel

Metabolic activity

The cytotoxicity of the GC nanogel was assessed by MTT assay using a set of cell lines; slightly different results were obtained with each one (Fig. 1). The control with culture medium diluted with 25% of water presents a lower cell growth as compared to the straight medium, an effect probably due to the dilution of the nutrients. It must be remarked that in none of the tested nanogel concentrations the mitochondrial activity was lower than the one observed at time zero, indicating that the GC

Discussion

Although GC is recognized as a highly biocompatible chitosan derivative (Carreño-Gómez and Duncan, 1997), a definitive statement on this matter requires further experimental evidences. Thus, the purpose of the present work was to perform a comprehensive study of the GC based-nanogel biocompatibility. The GC used for nanogel synthesis, purchase from Sigma–Aldrich, were thoroughly characterized in our previous work, due to the lack of consistent data in literature. The amphiphilic polymer

Conclusion

The biocompatibility of GC nanogels was comprehensively characterized. Although slightly reducing the growth rate of RAW and HMEC cell lines such an effect could not be explained by cell membrane compromising nor apoptosis/necrosis induction. A more severe effect observed in the case of RAW cells could be at least partially explained by the observed effect of cell cycle arrest; however for the HMEC this was not the case, an issue that remains to be clarified. It must be remarked that fairly

Conflict of Interest

The authors declare that there are no conflicts of interest.

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Acknowledgments

Paula Pereira thanks FCT, the Ph.D. grant ref SFRH/BD/64977/2009. This work was also supported by a grant from the Spanish Ministry of Economy and Competitivity (SAF2011-30337-C02-02). We also acknowledge the European Union Seventh Framework Programme [FP7/REGPOT-2012-2013.1] under grant agreement BIOCAPS-316265. MP acknowledges fellowship from Spanish Ministry of Education (FPU predoctoral grant program).

References (34)

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