Detailed toxicity evaluation of β-cyclodextrin coated iron oxide nanoparticles for biomedical applications

https://doi.org/10.1016/j.ijbiomac.2017.09.067Get rights and content

Abstract

The application of iron oxide nanoparticles [IONPs] in biomedical research is progressively increasing, leading to the rapid development of biocompatible and surface modified IONPs. However, there is still a need of information pertaining to its cellular and acute toxicity profile. This work reports the synthesis of β-cyclodextrin coated iron oxide nanoparticles (βCD-IONPs) and their characterization using spectroscopic (FT-IR), thermal (TGA) and surface analysis (TEM, SEM, BET and Zeta potential). All the characterization techniques displayed the synthesis of well dispersed, rod shaped βCD-IONPs of 45 nm. Time dependent cellular uptake of these nanoparticles was also evaluated using Prussian blue staining. Further, cytocompatibility analysis was executed in mouse fibroblast cell line (NIH 3T3) using MTT and LDH assays, respectively which did not show any cytotoxic indications of βCD-IONPs. Finally, acute toxicity analysis was carried out in female Wistar rats according to OECD guidelines 420. Rats were exposed to the highest dose (2000 mg/kg) of βCD-IONPs along with control and observed for 14 days. After two weeks of administration, tissues and blood were collected and subjected to histopathological and biochemical analysis (SGOT, SGPT and ALP). Animals were sacrificed and gross necropsy was carried out. It has been shown that βCD-IONPs does not have any significant toxic effect at the cellular level. Thus, this study provides new perspectives for future biomedical applications.

Introduction

Iron oxide nanoparticles (IONPs) have been used in various biomedical applications because of their unique physical and chemical properties. The most common applications include cellular labeling [1], [2] tracking [3], use as contrast agents in magnetic resonance imaging (MRI) [4], [5], [6] for therapeutic purpose in hyperthermia [7], [8] and drug delivery [9], [10].

In spite of these applications, IONPs have limited use in biomedical applications because of its hydrophobic surface, instability in aqueous media, prolonged retention time in the cells and its inherent toxicity. Employing particles which are toxic in nature can significantly affect the therapeutic efficacy of a cell based therapy [11] and can lead to morphological changes, mitochondrial impairment and membrane leakage [12]. These issues are not very substantial and can be overcome by proper surface modifications of the nanoparticles. It has been reported that surface functionalization significantly improves the stability, biocompatibility and overall shelf life of the nanoparticles [13]. A lot of work has been done to modify the IONPs surface till date. Various biocompatible coating agents which have been explored so far includes polymers such as poly(vinyl pyrrolidone) (PVP) [14] and poly(vinyl alcohol) (PVA) [15], polysaccharides such as dextran [16] and simple molecules such as citrate [17] and ascorbate [18], [19].

Cyclodextrins are cyclic oligosaccharides consisting of six (α-cyclodextrin), seven (β-cyclodextrin), eight (γ- cyclodextrin) or more glucopyranose units linked by α-(1  4) linkage. Out of all these, β-cyclodextrin is the most reported sugar molecule because of its easy access and inexpensive nature. The most important feature of cyclodextrins is their ability to form solid inclusion complexes with wide variety of guest molecules wherein, the guest molecule is held within the hydrophobic cavity of host cyclodextrin. Because of this property, β-cyclodextrin is widely used in pharmaceutical industries as a most common drug delivery agent. [20].

Iron oxide nanoparticles functionalized with β-cyclodextrin have been found to have number of biological applications. Banerjee and Chen have shown the loading and release of model hydrophobic drug ketoprofen from βCD-citrate grafted magnetic nanoparticles which served as nanocarrier [21]. Whereas, Li et al. have revealed the use of carboxy-methyl βCD coated IONPs for selective binding and detection of cholesterol crystals using MRI [22]. Polymer functionalized βCD-IONPs were used as drug delivery vehicle for the treatment of breast cancer which was reported by Yallapu et al. [23]. Recent work by Monteiro et al. have shown the synthesis of βCD-IONPs for irinotecan delivery as an anti-tumor treatment [24]. Besides biological applications, βCD coated IONPs have also been used for other industrial applications such as selective oxidation of alcohols [25], decontamination of U(VI) bearing effluents [26] and heavy metals removal from industrial waters [27].

For any successful cell based therapy, it is of utmost importance that the nanoparticles employed should be non-toxic. Keeping in mind the possible advantages of IONPs, there is still a need of suitable coating material with desired stability and biocompatibility. And further, to examine the potential cytotoxicity associated with these coating agents and IONPs. There are very few reports available displaying the toxicity profile of various β-cyclodextrin formulations. In one of the article, Kiss et al. have provided the cytotoxicity status of βCD derivatives on CACO-2 cell line using MTT assay [28]. Similarly, cytotoxicity profile of α-cyclodextrin derivatives were reported by Roka et al. [29] Acute toxicity studies have also been reported on rats using novel 2-hydroxypropyl-β-cyclodextrin [30] and β-cyclodextrin nanosponge formulations [31], respectively. Referring to these data, it can be easily stated that there is no literature available to best of our knowledge which depicts the detailed in vitro and in vivo toxicity profiling of β-cyclodextrin nanoparticles.

In this paper, we have explored various properties of β-cyclodextrin for functionalizing the IONPs surface and subsequently their cellular uptake and in vitro cytoxicity assays such as MTT and LDH for studying the toxic effects of these nanoparticles. In order to assess the cytotoxicity profile of βCD-IONPs, bare IONPs as well as commercial polyvinylpyrrolidone (PVP) coated IONPs were also incorporated for all the in vitro assays. Lastly, the highest concentration of βCD-IONPs (2000 mg/kg) was evaluated for in vivo acute toxicity study in Wistar rats for 14 days. Animals were sacrificed after the treatment and various tissues were subjected to histopathological evaluation. To evaluate the toxic effects of βCD-IONPs on liver, various biochemical tests such as Serum glutamic-oxaloacetic transaminase (SGOT), Serum glutamic pyruvate transaminase (SGPT) and Alkaline phosphatase (ALP) were carried out using serum.

Thus, the paper emphasizes on the synthesis of stable and biocompatible IONPs using β-cyclodextrin which can be subsequently used for various biomedical applications.

Section snippets

Materials and methods

Ferrous chloride tetrahydrate (FeCl2·4H2O), ferric chloride hexahydrate (FeCl3·6H2O) and β-cyclodextrin were procured from Sigma Aldrich, USA. Ammonium hydroxide was obtained from SDFCL, Mumbai, India. All the chemicals were of analytical grade and used without further purification. High glucose Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), Dulbecco’s Phosphate Buffered Saline (DPBS), 0.25% Trypsin-EDTA with phenol red indicator were acquired from Cell Clone, Genetix,

Results and discussion

Black colored βCD-IONPs were synthesized using co-precipitation method [32], [33]. The particles obtained were magnetic in nature which can be seen in Fig. 1b. Fig. 1a shows the representation of β-cyclodextrin coating over iron oxide nanoparticles. Particle characterization was carried out using FT-IR, SEM, BET, TEM, Zeta potential and TG analysis.

Conclusion

In this study, we have seen in detail the cellular toxic effects of βCD-IONPs in vitro as well as in vivo. β-cyclodextrin was used as a coating agent for the synthesis of stable and biocompatible iron oxide nanoparticles. Synthesized IONPs were characterized using FT-IR, TEM, SEM, Zeta potential, TGA and BET surface area. All the techniques confirmed the synthesis of well dispersed and stable rod shaped βCD-IONPs of the size 45 nm. Prussian blue staining also revealed complete uptake of IONPs

Acknowledgements

The authors would like to acknowledge Dept. of MEMS, IIT Bombay for BET surface area facility, Dr. K. C. Barick, Bhabha Atomic Research Centre (BARC) for TGA facility and Dr. Kolja Them, University Medical Center, Hamburg for TEM and SEM analysis.

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