Elsevier

Biomedicine & Pharmacotherapy

Volume 97, January 2018, Pages 26-37
Biomedicine & Pharmacotherapy

Original article
In vitro and in vivo toxicity assessment of alginate/eudragit S 100-enclosed chitosan–calcium phosphate-loaded iron saturated bovine lactoferrin nanocapsules (Fe-bLf NCs)

https://doi.org/10.1016/j.biopha.2017.10.121Get rights and content

Abstract

Lactoferrin has been known to have antimicrobial properties. This research was conducted to investigate the toxicity of Alginate/EUDRAGIT® S 100-enclosed chitosan–calcium phosphate-loaded Fe-bLf nanocapsules (NCs) by in vitro and in vivo assays. Brine shrimp lethality assay showed that the LC50 value of NCs was more than 1 mg/mL which indicated that NCs was not toxic to Brine shrimp. However, the LC50 values for the positive control potassium dichromate at 24 h is 64.15 μg/mL, which was demostrated the toxic effect against the brine shrimp. MTT cytotoxicity assay also revealed that NCs was not toxic against non-cancerous Vero cell line with IC50 values of 536 μg/mL. Genotoxicity studies by comet assay on Vero cells revealed that NCs exerted no significant genotoxic at 100 μg/mL without tail or shorter comet tail. Allium cepa root assay carried out at 125, 250, 500 and 1000 μg/mL for 24 h revealed that the NCs was destitute of significant genotoxic effect under experimental conditions. The results show that there is no significant difference (p > 0.05) in mitotic index between the deionized water and NCs treated Allium cepa root tip cells. In conclusion, no toxicity was observed in NCs in this study. Therefore, nontoxic NCs has the good potential to develop as a therapeutic agent.

Introduction

Nanotechnology is a speedily rising area having potential applications in many fields. For the past few decades various formulations of nanoparticles have been used for drug delivery research to increase therapeutic benefit [1]. Up to now, nanoparticles (NPs) have been prepared from metal and non-metal, polymeric materials and bioceramics [2]. Due to their unique physicochemical and electrical properties, nano-sized materials have gained substantial attraction in the field of electronics, biotechnology, and aerospace engineering [2]. In the field of medicine NPs are being used as a novel delivery system for drugs, proteins, DNA, and monoclonal antibodies [3], [4], [5]. Key components in the nanoparticle preparation may help to enhance the therapeutic properties of nanoproducts. Alginate is a pH sensitive FDA approved polymer which protects drugs in the low acidic pH conditions inside the stomach and able to release drugs in the alkaline pH in the intestinal regions where all the absorption occurs [6], [7]. Eudragit S 100 and chitosan polymers are mucoadhesive and able to stick to the intestinal walls and help in endocytosis and exocytosis through the intestine to the main blood streams for further distribution of drug molecules in the different parts of the body [8], [9], [10]. Nevertheless, many challenges must be overcome if the application of nanotechnology is to increase therapeutic benefit and yield improved therapies. Research has established that contact to these combustion derived ultrafine particles/nanoparticles is associated with a wide variety of effects [11]. In view of the potential applications of NPs in many fields and the rising worries of FDA about the toxic potential of nanoproducts, it is the need of the hour to study the toxicological effects of nanoproducts. In this study for the first time the toxicity of alginate-enclosed chitosan-calcium phosphate-loaded Fe-bovine lactoferrin nanocapsules (NCs) was reported by using brine shrimp lethality assay, Allium cepa assay, comet assay and cell cytotoxicity assay, especially in case of genotoxicity.

In vivo techniques includes Allium cepa assay and brine shrimp lethality assay [12] was used in this study. However, techniques to detect in vitro cytotoxicity involve the use of comet assay, and tissue culture [13] methods were also used in this study. The cytotoxicity of NCs was conducted using Vero cell, a non-cancerous cell to observe cytotoxicity of NCs in a cellular model. Subsequently, the comet assay was conducted to evaluate in vitro genotoxicity of NCs. Higher plants such as Allium cepa are good genetic models to recognise genotoxicity such as chromosome aberrations and changes in the mitotic cycle of NCs. Therefore, the current study utilized the root tips cells of Allium cepa for evaluating the genotoxic effects of NCs.

Section snippets

Preparation of endotoxin-Free Fe-bLf

Endotoxin-free bovine lactoferrin (bLf) was prepared from Australian bovine milk [14]. Endotoxin was evaluated by using Genscript ToxinSensor™ (NJ, USA), Chromogenic Limulus Amebocyte Lysate Endotoxin Assay Kit (Genscript ToxinSensor). Ferum-bLf was produced from bLf based on the previously described method established in Deakin’s laboratory, Australia. Following treatment with mild acidic solution (pH 2.6), bLf was dialyzed for a duration of 48 h in 0.1 M citric acid to remove bound metal ions

Preparation and characterization of NCs

Dynamic light scattering (DLS) spectrometric results indicate an increase in the hydrodynamic size of NCs after coating with chitosan and further encapsulation using alginate. The physio-chemical characteristics of the polymeric NCs revealed void (protein free) NCs had a zeta potential negative charge of −2.6 ± 0.6 mV and a dispersity index of 0.231 ± 0.05. Fe-bLf-NCs showed similar results to void NCs where NC were spherical in structure, had a negative zeta potential charge of −2.7 + 0.4 mV and poly

Preparation and characterization of NCs

Even though substantial developments in drug delivery, the capability to release drugs at a definite location in the body in a controlled manner remains unsettled. Smart chemistry of polymer-based drug delivery systems that can alter nanocarriers polymer structural design and release their load consequent to an external stimulus is thus attaining growing reputation [10]. Polymer-based nanocarrier systems that comprise an exclusive pH-sensitive modality undertake destabilization or decomposition

Conclusion

The results from cytotoxicity test demonstrated that NCs was non cytotoxic against the Vero cells and A. salina. The A. cepa test and comet assay demonstrated that NCs exhibited no significant genotoxicity, which is substantial for future investigation for human applications. In conclusion, the results in in vitro and in vivo tests demonstrated that NCs was devoid of significant toxic effect under our experimental conditions. For further future studies, a detail in vivo toxicity studies can be

Conflict of interest

All authors have been no potential conflict of interest.

Acknowledgements

The authors duly acknowledge Dr KK Sishir for the preparation of NCs used in this study.

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