Elsevier

Applied Surface Science

Volume 470, 15 March 2019, Pages 744-754
Applied Surface Science

Full Length Article
Oxidized and amino-functionalized nanodiamonds as shuttle for delivery of plant secondary metabolites: Interplay between chemical affinity and bioactivity

https://doi.org/10.1016/j.apsusc.2018.11.161Get rights and content

Highlights

  • Engineering and functionalization of detonation nanodiamonds (DND) surfaces.

  • Investigation of surface chemistry, morphology and structure of modified DND.

  • Adducts of DND with plant metabolites characterized by anticancer activity.

  • Relationship between DND surface engineering and tumoral cell viability.

Abstract

This study is focused on the surface engineering of detonation nanodiamonds (DND) for the delivering of therapeutic agents, and on the developing of strategies for the DND conjugation with biologically active substances. Oxidized and differently amino-functionalized DND have been investigated to identify their chemical and structural features. A series of complexes have been produced by reacting the activated DND particles with four antioxidant molecules abundant in grapes and wine: resveratrol (R), rutin hydrate (U), epicatechin (E) and chlorogenic acid (C). Drug up-take is found strictly dependent on the affinity between molecules and selectively modified DND surfaces. All adducts, prepared avoiding potentially toxic solvents, reached a concentration suitable for therapeutic applications. MTT assay, performed on SH-SY5Y neuroblastoma cell line, evidenced that the conjugation with DND amplifies the bioactivity of the metabolites with respect to the relative pure molecules. However, depending on the DND surface chemistry, the various metabolites are found to exert markedly different effects on tumor cell viability, producing increase or decrease of proliferation. These findings indicate that a tailoring of the DND surface chemistry, specific for each molecule, is required not only to improve binding/delivering of active substances to biological targets, but also to achieve the desired therapeutic results.

Introduction

Among the large spectrum of nanoparticles recently investigated as shuttle for drug delivery, nanodiamonds have emerged as very promising platforms. The multitude of beneficial properties make the nano sized sp3-coordinated carbons, and in particular detonation nanodiamond (DND) [1], [2] a material suitable to realize, at least partially, the concept of magic bullet (i.e. a system to selectively carry and delivery therapeutics), anticipated by P. Ehrlich at the beginning of the XX century. DND is a large-scale product obtained from detonation of trinitrotoluene and consists of nanograins, sized from 5 to 10 nm, formed by a diamond core surrounded by a graphitic shell [3], [4].

This material not only bares the well-known bulk diamond properties, like hardness, mechanical resistance and stiffness, but also possesses several attractive properties related to the nanosized dimensionssuch as: confinement effects, unusual values of surface energy and high surface area (up to 300 m2/g) [5]. Nowadays, DND is finding applications in many technological fields, from nanoelectronics to nanocatalysis [6], [7], [8], [9], [10], [11], [12], [13].

An ever-increasing field of interest is that of bio-related applications and nanomedicine, where diamond nanostructures are proposed for a series of different purposes, from scaffold for tissue-growth and site-specific molecular targeting/labeling [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24]. Two main peculiarities that distinguish DND from other nanostructures are the extremely tuneable surface chemistry and the high biocompatibility.

As regards the first item, nanodiamonds represent a platform capable of a wide variety of functionalizations [25], [26]. Regarding the biocompatibility, several studies have been performed on the interaction between DND and cells, evidencing that, compared to other carbon forms, this nanomaterial is characterized by the lowest cytotoxicity for a large variety of cell lines. These nanostructure can be indeed internalized by cells via endocytosis, without interfering with cell viability, even at high concentrations [27], [28], [29], [30].

Such abilities suggested the use of DND as an excellent shuttle, and it is not surprising that DND has been conjugated to a wide variety of biological moieties such as drugs, proteins, antibodies, DNA and siRNA for several in vitro and in vivo experiments [31], [32], [33], [34], [35], [36], [37], [38]. On the basis of several positive clinical studies, nanodiamond systems have been included in the design of de-risked drug development platform technology, called Phenotypic Personalized MedicineDrug Development (PPM-DD), a new approach based on the optimization of therapeutic combinations [39].

In the field of drug delivery our research is focussed on the preparation of adducts coupling DND and natural substances for applications in cancer therapy. It is well known that some natural extracts can act as growth inhibitors and treatment adjuvants in cancer therapy or chemotherapeutic lines [40], [41]. However, therapies by natural antioxidant often suffer of some limitations. First of all, due to the low reduction potential, natural antioxidants are in general characterized by a scarce chemical stability and are thus easily oxidized in culture media, losing their therapeutic effect. Moreover, frequently the drug solubility in water is not sufficient to reach an active therapeutic concentration, and is therefore needed to use organic solvents, that unavoidably increase the whole therapy toxicity. As regards the employing of antioxidants coupled with nanosized carriers, the nanostructures must not only avoid drug degradation and guarantee good performances in drug-loading/releasing processes, but should also not interfere negatively with the catabolic pathway of the active molecules. On such basis, DND-based vectors are almost the only ones able to match all the bio requirements [42].

In recent papers we reported about nanodiamonds conjugated with two plant compounds, 5,7-dimethoxycoumarin and quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one), and highlighted the antiproliferative and differentiative action of such adducts against HeLa [43] and B16-F10 [44] cells. In particular, it was found that the control of DND surface, obtained via specific chemical/physical strategies, enabled not only to tune the affinity for the drugs but also to drive the intrinsic bioactivity of the drugs. The results evidenced a relationship connecting chemical composition and polarity of DND surfaces with the biological activity of plant secondary metabolites. The output of such investigations triggered further researches aimed at exploring different functionalities and at understanding how the DND chemistry influences the up-take of various molecules and their biological effects.

In this view, differently terminated DND surfaces have been now generated by oxidation and amidation processes. Oxidized DND (DND-Ox), already tested in a previous research [43] have been proven to be a successful system for the delivery of a number of therapeutics [45]. On the other hand NH2-terminated DND, a versatile material for a series of advanced applications [46], is attracting a lot of attention for its performances in several bio-related areas, from the promotion of neuronal cell culture [47], [48] to delivery of drugs [49] and gene therapy [50], [51], [33].

As regards the amino-terminated DND (AT-DND), in the present research three different types of samples were obtained using ethylenediamine, 1,3-propylenediamine and 1,6-hexamethylenediamine, respectively.

Deep investigations were carried out by means of TEM, FT-IR, XPS and Raman spectroscopy to evidence the chemical/structural features and the surface polarity of pristine and treated DND.

The DND systems formulated following different procedures were conjugated with four metabolites characterized by a high level of bioactivity: the water-soluble rutin hydrate (U), poorly soluble resveratrol (R) and epicatechin (E) or partially soluble chlorogenic acid (C). Pure molecules and DND-based adducts were tested against neuroblastoma SH-SY5Y cell lines. MTT assay was performed to compare the effect on tumor cell growth of the plant metabolites conjugated with the various functionalized DNDs and of their pure forms. Results indicate that the chemical state of the DND surface plays a key role in modulating the biological effects of R, U, E and C compounds, and imply the need to formulate specific DND-based systems for each active molecule.

Section snippets

DND processing

Organic reagents and dry solvents stored over a molecular sieve were purchased by Sigma-Aldrich and used as received.

For the present experiments we used DND powders provided by International Technology Center (ITC, USA). In order to remove amorphous carbonaceous phases, to enhance the presence of oxygen-containing groups on the DND surface and to disintegrate extremely tight diamond aggregates, treatments that combined strong oxidation with power ultrasound were performed. The as-received DND

Analysis of processed DND samples

Fig. 3 shows typical TEM images of pristine (DND), oxidized (DND-Ox) and amino-terminated DND powders (DND-Et, DND-Pro and DND-Hex).

For the pristine DND sample the TEM analysis evidences the presence of rather large (some tens of nanometers) and tightly agglomerated grains, produced by the strong tendency to self-assemble typical of detonation nanodiamond particles. For the processed powders, we can observe that the samples consist of much more disaggregated entities with a highly regular

Conclusions

The aim of the present research was to study the correlation between the DND surface chemical properties, that are known to drive the conjugation with molecules, and the modulation of the bioactivity of such compounds. To enable a look into this complex issue, nanodiamond samples produced with very dissimilar surface chemistry have been conjugated with R, U, E and C plant secondary metabolites typical of grape fruits and wine and tested against SH-SY5Y cell line.

A first achievement of this

Acknowledgements

RC wishes to gratefully acknowledge FBK (Fondazione Bruno Kessler) for financial support.

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    Both R.C. and V.N. contributed equally to this work.

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