Human nitric oxide biomarker as potential NO donor in conjunction with superparamagnetic iron oxide @ gold core shell nanoparticles for cancer therapeutics

https://doi.org/10.1016/j.colsurfb.2017.12.052Get rights and content

Highlights

  • N-Nitrosothioproline (NTHP) conjugated magnetic nanoparticles for cancer therapeutics.

  • NTHP, a non-toxic human biomarker shows enhanced NO release with light.

  • Nano carriers shows excellent cancer cell inhibition in pM range compared to control.

  • May prove promising Nano carriers for cancer photodynamic therapy.

  • Clinical trials may lead to potential cancer therapeutic with minimum side effects.

Abstract

Nitric oxide releasing superparamagnetic (Fe3O4-Au@NTHP) nanoparticles were synthesized by conjugation of human biomarker of nitric oxide, N-nitrosothioproline with iron oxide-gold (Fe3O4-Au) core shell nanoparticles. The structure and morphology of the prepared nanoparticles were confirmed by ATR-FTIR, HR-TEM, EDAX, XPS, DLS and VSM measurements. N-nitrosothioproline is a natural molecule and nontoxic to humans. Thus, the core shell nanoparticles prepared were highly biocompatible. The prepared Fe3O4-Au@NTHP nanoparticles also provided an excellent release of nitric oxide in dark and upon light irradiation for cancer treatment. The amount of NO release was controllable with the wavelength of light and time of irradiation. The developed nanoparticles provided efficient cellular uptake and good cytotoxicity in picomolar range when tested on HeLa cancerous cells. These nanoparticles on account of their controllable NO release can also be used to release small amount of NO for killing cancerous cells without any toxic effect. Furthermore, the magnetic and photochemical properties of these nanoparticles provides dual platform for magneto therapy and phototherapy for cancer treatment.

Graphical abstract

Magnetic core shell nanoparticles with conjugated N-nitrosothiproline (a human biomarker of nitric oxide) for photodyanmic cancer therapy with controlled nitric oxide releasing capability using different wavelengths of light.

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Introduction

Nitric oxide (NO) is an important biomolecule affecting wide range of physiological properties. NO is a free radical signalling molecule having antimicrobial [1] and anticancer properties [[2], [3], [4]]. Thus, NO is an important pharmaceutical agent because of its effectiveness to kill tumour cells. However, the anticancer nature of NO depends on its concentration and dosage. It can be cytotoxic at certain concentration against various pathogens and cancerous tumour and at the same time can act as a mediator in cancer phenotypes at higher concentration [5]. NO is highly unstable molecule and requires additional means to stabilize it for effective anticancer delivery at specific tumour site [6].

Thus, to control the stable delivery of NO at specific level, several nano carriers have been reported. Some of them include transition metal nitrosyl complexes, [7] polymeric nanoparticles, [8] xerogels, [9] silica nanoparticles [10] and gold nanoparticles (Au NPs) [11]. Au NPs because of their small size and dense core have been reported to accumulate successfully in tumour tissues and can serve as an excellent delivery vehicle for NO [12]. Au NPs when bonded with thiol groups like 2-mercapto-5-nitro benzimidazole has the potential to release NO for killing the cancer cells [13]. Additionally, light alters the rate of NO release as an external activator for photoactivable NO donors [14]. Thus, Au NPs because of their photothermic effect has emerged as potential anticancer agents.

Surprisingly, there are few reports where iron oxide nanoparticles (IONPs) have been used as NO releasing agents for cancer treatment [15,16]. IONPs have the potential to generate localised heat on applying an external magnetic field to the targeted site and thus can attract drug loaded nanoparticles from blood circulation as cancerous cells are more heat sensitive than normal cells [17]. To make IONPs more stable and biocompatible with propensity for surface modification, the IONPs has been stabilized by gold shell, generating Fe3O4-Au core shell NPs. The Fe3O4-Au core shell NPs have proved to be an excellent drug carrier and probes. Moreover, the Au shell on account of its high bonding efficiency with thiols makes them an ideal platform in designing of nanodrug carriers with desired properties. The Fe3O4-Au core shell particles have been effectively reported as contrast agents for Computed Tomography [18] (CT), magnetic resonance imaging [19] (MRI) and targeted drug delivery with application of magnetic field [20,21]. The modified core shell nanoparticles retains magnetic properties of iron and high biocompatibility of Au in addition to exhibiting high propensity for cancer specific modifications. The core shell nanoparticles thus serve as dual role of magnetic and photo thermal agents [22].

For designing the NO releasing agents based on the Fe3O4-Au core shell NPs, care must be taken in selecting non-toxic NO releasing agents as most of the NO releasing agents or their intermediates are toxic in nature which can dilutes their potential in cancer therapeutic effect [6,23].

Thiazolidine-4-carboxylic acid commonly called as thioproline (THP) is a cyclic sulphur containing amino acid reported to have anticancer properties though, the claim is disputed. Additionally, THP has also been investigated on rats for inhibiting the stomach cancer originated by nitrite and benzylmethylamine [24,25].

Nevertheless, THP has many important biological properties which make it an ideal molecule of biological importance. It has been reported that THP protects rat liver against hepatotoxic effect of ethanol, carbon tetrachloride, acetoaminophen and thiourea [26]. The THP on account of its medicinal importance has been clinically used to treat liver disease and gastrointestinal disturbances from past 20 years [27].

Besides, one of the most important properties of THP is its capability to detoxify nitroso compounds and nitrites and thus acting as a sensitive probe for evaluating nitrosating capacity [28,29]. THP is also known as efficient nitrite trapping agents in-vitro and in-vivo for nitrosating species in acidic condition and has been explored widely in various therapeutic effects [30,31]. The nitrosamine formed by the reaction of THP with nitrite ions/NO is non-toxic and is excreted un-metabolised as N-nitrothiazolidine carboxylic acid, also known as N-nitrosothioproline (NTHP) in the urine. Thus, NTHP acts as a biomarker for human nitrite concentrations. Recently, we have reported the potential of THP for electrochemical detection of nitrite ions in micromolar range with good efficiency [29].

Thus in the present work we took the advantage of biological molecule NTHP, its Au binding properties, and coated the Fe3O4-Au magnetic core-shell nanoparticles with NTHP using self-assembly. The hybrid core shell nanoparticles proved to be an effective NO releasing agents under physiological conditions. The NTHP being non-toxic to humans provided an efficient NO releasing agent having cytotoxicity towards cancerous cell lines. The developed nanocarriers proved to be effective in killing cancer cell lines (HeLa) with anticancer activity being higher under light irradiation making them compatible with photo thermal treatment. Further, these core shell nanoparticles releases different amount of NO under different wavelength of lights making them an excellent carriers for controlled release of NO at tumour site.

To the best of our knowledge, this work is a first of its kind where magnetic core-shell nanoparticles were coated with the nature’s biomarker NTHP for releasing NO in cancer therapeutics. The proposed NPs have high practical potential to be used in cancer theranostics with minimal or no side effects on the normal cells. Furthermore, the developed NO releasing core shell NPs can be an ideal substrate for magnetically guided cancer therapeutics and imaging (FMRI) in addition to the photo thermal therapy.

Section snippets

Reagents and solution

Chloroauric acid (HAuCl4), L-4-thiazolidine-4-carboxylic acid and Trifluoroacetic acid (TFA) were purchased from Sigma Aldrich (St. Louis, MO, USA). Tri-sodium citrate, sodium nitrite (NaNO2), Iron (III) chloride (FeCl3), Iron (II) Sulphate (FeSO4·7H2O), Acetonitrile (ACN) HPLC grade, HCl (0.1N) and NaOH to maintain the pH, were purchased from Finar (India) and were used without further purification.

Preparation of iron oxide nanoparticles

Iron Oxide nanoparticles (Fe3O4) (IONPs) were prepared by the chemical co-precipitation method [

Characterization of synthesized nanoparticles

The N-nitrosothioproline coated superparamagnetic Fe3O4-Au core shell NPs (Fe3O4-Au@NTHP) were prepared as shown in Scheme 1.

Briefly, Fe3O4-Au core-shell NPs were coated with the self-assembled monolayer of THP (Scheme 1A) and the resultant Fe3O4-Au@THP core-shell NPs were reacted with sodium nitrite to form Fe3O4-Au@NTHP core shell NPs (Scheme 1B).

The UV–vis absorption spectrum of IONPs shows λ max at 224 nm due to d–d transitions as shown in Fig. 1A. This was in agreement with the reports,

Conclusion

In summary, we have prepared superparamagnetic Fe3O4-Au@NTHP core shell nanoparticles that can be photo-cleaved to release NO at desired concentrations for cancer therapeutics. These core shell nanoparticles were prepared using nature’s biomarker NTHP which formed self-assembled coatings on the Fe3O4-Au core shell nanoparticles. The nanoparticles were characterised for surface chemistry and morphology using, HRTEM, XPS and ATR-FTIR. The particles show superparamagnetic behaviour as revealed by

Acknowledgements

This work was made possible by a grant from the SVNIT, Surat and DST (YSS/2015/00184) financial support for project. We would like to thanks MNIT, Jaipur and Charusat University for providing us HRTEM, XPS and VSM facility and also Genexplore for carrying out the biological study.

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