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TurboBeads are highly magnetic cobalt nanoparticles with diameters of below 50 nm. The surface of the particles is coated with a graphene shell, which is covalently functionalised with primary aliphatic azido or amino groups (≥0.1 mmol/g).²²³

Figure 1.56. A schematisation of the structures of TurboBeads: left, Click; right, amine coated

Their strong paramagnetism has been exploited for the development of a breakthrough purification technique, which allows the magnetic separation of chemicals of various nature from a reaction mixture as well as bulk samples. In fact, quick and efficacious separation of TurboBeads-conjugates from other chemical species can be achieved by using a laboratory-scale neodymium magnet. The magnet is put on the outside of the flask containing the sample and attracts the NP-conjugates, which

94 gather in its proximity. As NP-conjugates can’t be removed from this position, other species present in the reaction mixture can be simply washed away. Also other kinds of paramagnetic nanoparticles (such as SPIONs, see §1.2.3a) can undergo magnetic separation, but in the case of TurboBeads this process is particularly fast and efficient.

This novel kind of cobalt nanoparticles, on the market since 2007, is being applied in an increasing number of science fields: for instance, they play an important role in biochemical assays when bound to targeting moieties of biomolecular interest.²²⁴ Moreover, they are increasingly gaining in importance for what concerns organic synthesis, as they can be further functionalised with expensive catalysts to improve their recovery from the reaction mixture, in order to recycle them. In addition, if functionalised with heavy metal chelators, they constitute very useful tools in clinical and environmental toxicology.

For what concerns theranostic applications, the use of TurboBeads or, more in general, of cobalt nanoparticles (CoNPs) is currently being the object of many pharmacological studies. Thanks to their elevated magnetic moment, CoNPs could be the ideal candidate for magnetic field assisted targeted drug delivery or hyperthermia. Actually, it has been observed that CoNPs’ magnetic moment is twice as high as that of SPIONs,²²⁵ property which could render them suitable for applications in magnetic resonance imaging. For instance, they could replace SPIONs-based contrast media in the diagnosis of hidden, scarcely detectable tumors. Furthermore, being the metal with the highest Curie point, cobalt firmly maintains its magnetic properties also in stress conditions: this could allow the development of nanosystems characterised by a very prolonged shelf-life.²²⁶

At any rate, the development of in vivo applicable CoNPs-based theranostic systems is being held back by non negligible toxicological issues. If SPIONs’ ROS-mediated toxicity still has to be unanimously acknowledged, it is commonly known that cobalt may induce oxidative stress in vivo:²¹³ metallic cobalt can, in fact, be oxidated to generate various oxidation state cobalt ions. This newly generated reactive species can in turn be reduced again, hence cobalt’s oxidizing activity. Recently, it has been observed by Jiang et al. that CoNPs exposure induces cytotoxicity and genotoxicity in human T cells in vitro.²²⁷ Moreover, CoNPs have a remarkable ability to permeate membranes²²⁸ and they can be easily accumulated by cells and exert their oxidative DNA damage activity. However, CoNPs toxicity may be fine-tuned by the application of a suitable coating which could limit their systemic oxidative activity; this relevant cytotoxic activity could be even addressed towards tumor cells by means of the already described targeting techniques. The modulation of CoNPs’ toxicity by the addition of a protective coating seems however to constitute the most promising strategy: it has been observed, for instance, that CoNPs coated with graphitic shells (feature presented also by TurboBeads) can be applied in hyperthermia therapy to generate thermally localized cellular damage, which can induce cell death and necrosis in cancerous tissue.²²⁹

Our work thus consisted in the functionalisation of cobalt TurboBeads, by means of copper (I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction or amidation reaction, with the previously described combretastatin A-4 alkyne derivative (57) or NHS-activated derivative (57b), in order to achieve a new, promising, theranostic CoNPs-based system.

Synthesis and characterisation of CoNPs-CA4 conjugate

95 The functionalisation of cobalt TurboBeads Click was achieved by carrying out an adapted version of copper-catalyzed azide-alkyne cycloaddition, or CuAAC, described by Sharpless et al.¹⁹⁷ In this reaction, the azide reacts specifically with the alkyne to give a triazole, as discussed in § 1.2.3.

To functionalise azide-coated CoNPs with combretastatin A-4 hexynoate (57), we activated the nanoparticles by washing them in dry toluene and sonicating for 10’ at r.t. for 3 times. This procedure consented a fine particle dispersion, which facilitated access to the azido functions on the NPs’

coating. Once washed, CoNPs were suspended in dry toluene under a N2 atmosphere and combretastatin hexynoate (57) (10 equiv.), CuI (0.5 equiv.) and triethylamine (1 equiv.) were added.

As TurboBeads Click’s azide loading is known (0.1 mmol azide/g), other reagents could be added in molar ratio instead of weight ratio, as it was for SPIONs. The suspension was sonicated for 16 h. at r.t.

under N2, then the nanoparticles were retrieved from the reaction mixture by magnetic separation and solvent was eliminated. The solid residue was washed with fresh toluene, then residual solvent was eliminated under vacuum to afford NP-conjugate (70) (Scheme 1.51).

Scheme 1.51.

The functionalisation of cobalt amine-coated TurboBeads was achieved by carrying out a preliminary activation of the carboxylic derivative of Combretastatin (35) to a reactive NHS-ester compound (57b), followed by an amidation reaction. This strategy has been widely used to bind to proteins, peptides, DNA fragments or customized linking chemicals utilizing EDC/NHS chemistry.

In details, to functionalise amine-coated CoNPs with combretastatin A-4 derivative (57b), we activated the nanoparticles by washing them in a freshly prepare Tris buffer and sonicating for 10’ at r.t. for 3 times. After these preliminary ones, we performed two more washing with a freshly prepared PBS buffer, as suggested on the website of Turbobeads company.The amidation reaction was thus carried out in this solvent that suspended either compound 57b and CoNPs. The suspension was sonicated for 2 h at r.t. under N2, then the nanoparticles were retrieved from the reaction mixture by

96 magnetic separation and solvent was eliminated. The solid residue was washed with fresh PBS followed by DCM, then residual solvent was eliminated under vacuum to afford NP-conjugate (70b) (Scheme 1.51).

NP-CA4 conjugate (70) has been analysed by means of: a) IR spectroscopy and b) thermogravimetric analysis.

a) FT-IR control of the reaction was particularly appropriated, as azide N=N=N stretching band could be found at 2090 cm^{-1 (}Figure 1.57, left). While the IR spectrum of blank TurboBeads is characterised by the presence of the azide stretching band, adduct 70’s spectrum (Figure 1.57, right) did not present any signal in that zone. This may be a convincing proof that functionalisation has successfully taken place.

Figure 1.57. Left: IR spectra of blank TurboBeads. Right: IR spectra of CoNPs-CA4 conjugate 70

b) TGA. For what concerns TGA analysis, a thermogram of CoNPs-CA4 conjugate (70) has been recorded and compared to that of blank TurboBeads (Figure 1.58). In the thermogram of the blank nanoparticles, a first, non-oxidative 3.8% weight loss is observed from 30°C to 700°C, under a nitrogen atmosphere.

The slope of the weight loss function brusquely increases as oxygen is inserted, leading to a further 1.45% weight loss. Therefore, a total 5.2% weight loss has been recorded, due to the thermal and oxidative degradation of NPs’ graphene coating. From 770°C a 2% weight increase is observed. This is presumably due to the oxidation of the metal cobalt core to cobalt oxides, once exposed to the oxygen-rich atmosphere after the degradation of the protective organic shell.

97

Figure 1.58. Left:Thermogram of blank TurboBeads. Right: thermogram of conjugate (70)

Adduct 70’s thermogram showed instead an interesting phenomenon. From 30°C to 700°C, under a N2

atmosphere, a 5,9% weight loss has been observed. As the non-oxidative weight loss was greater in the case of the adduct (5.9% against 3.8% of the blank) it could be inferred that the addition of an organic, thermodegradable portion on nanoparticles has occurred. The non-oxidative weight loss of adduct 70 was even greater than the total weight loss of the blank, both oxidative and not. At any rate, as oxygen was inserted, a sudden weight increase, amounting to 21%, was observed: this weight increase wass much more relevant than the one recorded in the case of the blank. Instead of showing a first oxidative weight loss and, only afterwards, a weight gain as in the case of the blank, adduct 70 just underwent a weight increase (Figure 1.58, right).

This led us to suppose that the prolonged sonication might have damaged the graphene shell on the NPs coating: in this case, cobalt would have been exposed to the oxidizing atmosphere from the very moment of oxygen insertion. However, an external coating damage would have seriously compromised the final achievement of the desired system, so we studied this phenomenon further. In order to verify whether sonication had actually affected the integrity of the graphene coating, we sonicated a sample of blank TurboBeads for 16 h. in the same conditions described in the synthesis of product (70). This new sample was then analysed by TGA (Figure 1.59).

98 Figure 1.59. Thermogram of sonicated blank compared with that of the first blank.

The thermograms of the first blank and the one of the sonicated blank were absolutely comparable, from which we can infer that sonication did not disrupt NPs’ coating. The weight gain observed in 70’s thermogram may thus be attributed to other phenomenons which do not invalidate the achievement of the final conjugate system. Moreover, a similar TGA weight increase in oxidizing atmosphere has also been reported for other kinds of commercial TurboBeads.²³⁰ For all these reasons, we suppose that the functionalisation of TurboBeads has successfully occurred.

Characterization of adduct 70b is still in progress and it is not reported here.

1.2.3c. Synthesis of combretastatin A-4-loaded gold nanoparticles