Therapeutic multifunctional carbon and 2D nanomaterials

1. Carbon nanotubes

The application of carbon nanotubes (CNTs) in nanomedicine has been widely explored thanks to their unique physicochemical properties. To allow fully exploiting their properties and enhancing their biocompatibility, functionalisation of their surface is a crucial step. In particular, the multifunctionalisation of CNTs is necessary to impart multimodalities for the development of future CNT-based multipotent therapeutic constructs (1). In this context, we developed an original and effective strategy for the covalent triple functionalisation of CNTs based on an arylation reaction performed in one step in the presence of a mixture of three aryldiazonium salts (2). The CNTs were functionalized with benzylamine moieties blocked with three different protecting groups that can be selectively and sequentially removed under specific conditions (Figure 1). This is the only approach developed to date for the covalent triple functionalization of CNTs. We exploited this strategy to control the covalent conjugation of an anticancer agent (gemcitabine), a targeting ligand of cancer cells (folic acid), and a fluorophore (fluorescein) (Figure 1) (3).

CNTs are also considered efficient carriers for the delivery of genetic material in vitro and in vivo. In this context, we functionalized CNTs with a series of dendrons of first and second generation bearing positive charges at their termini for siRNA complexation (4). The demonstration of the cellular uptake capacity, the low cytotoxicity, and the ability of the cationic CNT conjugates to silence cytotoxic genes suggests them as promising carriers for genetic material.

In addition, we investigated the functionalisation of CNTs with nucleobases for different applications (5). For instance, we functionalized CNTs with adenine via different linkers (hydrophilic or hydrophobic) to trigger the formation of catalytic silver nanoparticles with controlled sizes through metal/adenine coordination (6). These hybrids were employed as heterogeneous catalysts to promote the oxidation of hydroquinones to benzoquinones, an important class of molecules with antioxidant, anti-inflammatory, and anticancer activities. The nucleobase-CNT conjugates could also offer interesting opportunities as new biosensors or for DNA sorting through base pairing and/or other H-bonding combinations.

We also functionalized CNTs with multiple copies of a GM3-lactone mimetic antigen (7). The conjugate was able to efficiently interfere with metastatic-related events in terms of adhesion, migration and invasiveness, mediated by the antigen mimetic. The functionalized CNTs guaranteed an appropriate spatial arrangement of the mimetic allowing a stronger inhibition of migration and invasiveness of human melanoma cells compared to other multivalent constructs reported before.

1. Dinesh, B. et al. (2016) Nanoscale 8, 18596-18611.
2. Ménard-Moyon, C. et al. (2011) Chem. Eur. J. 17, 3222-3227.
3. Ménard-Moyon, C. et al. (2015) Chem. Eur. J. 21, 14886-14892.
4. Battigelli, A. et al. (2013) Small 9, 3610-3619.
5. a) Singh, P. et al. (2012). Carbon 50, 3170-3177. b) Singh, P. et al. (2012) Nanoscale 21, 1972-1974.
6. Singh, P. et al. (2011) Angew. Chem. Int. Ed. Engl. 50, 9893-9897. 7. Arosio P. et al. (2018) Org. Biomol. Chem. 16, 6086-6095.

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2. Graphene and 2D materials

Graphene oxide (GO) is an attractive nanomaterial for many applications. Controlling the functionalisation of GO is essential for the design of graphene-based conjugates with novel properties. Due to the high reactivity of the oxygenated moieties, mainly epoxy, hydroxyl, and carboxyl groups, several derivatization reactions may occur concomitantly. The reactivity of GO with amine derivatives has been exploited in the literature to design graphene-based conjugates, mainly through amidation. We undoubtedly demonstrate using magic angle spinning solid-state NMR that the reaction between GO and amine functions occurs via ring opening of the epoxides, and not by amidation (Figure 2) (1). We also proved that there is a negligible amount of carboxylic acid groups in two GO samples obtained by a different synthesis process, hence eliminating the possibility of amidation reactions with amine derivatives.

GO is constituted of various oxygen-containing functionalities, primarily epoxides and hydroxyl groups on the basal plane, with a very low amount of carbonyl, quinone, carboxylic acid, phenol, and lactone functions at the edges. The high chemical reactivity of these oxygenated groups makes functionalisation difficult to control. We have investigated the reactivity of GO towards orthogonal reactions to selectively functionalise the hydroxyl groups. We explored both the esterification and the Williamson reaction (2). Our strategies presented the main advantage to occur in mild conditions, thus preserving the intrinsic properties of GO, whereas most reactions reported require relatively strong conditions, leading to partial reduction, and/or are not chemoselective. We have also extended our study to the ketones and examined their derivatization by the Wittig reaction. Our work leads to a better understanding of the reactivity of GO for controlled derivatization.

Double functionalization is a key aspect in the design of multifunctional GO with combined imaging, targeting, and therapeutic properties. In this context, we examined the combination of the nucleophilic epoxide ring opening with the derivatization of the hydroxyl groups through esterification or Williamson reaction (Figure 3a) (3). The most efficient method appeared to be the combination of the opening of the epoxides and the esterification of the hydroxyls. The conditions were selective and mild, thus preserving the structure of GO. The presence of amine functions allows for further derivatization with molecules of interest under mild conditions, for example, amidation.

In an alternative approach, two functional groups were covalently linked to GO in two steps: the first group was attached by an epoxide ring-opening reaction with a thiol-containing molecule and the second, bearing an amine function, was covalently conjugated to benzoquinone attached to the GO (Figure 3b). This method is straightforward and the reaction conditions are mild, allowing preservation of the structure and properties of GO.

The different strategies of double functionalisation of GO open promising perspectives to design graphene-based nanomaterials endowed of multiple properties.

One of the most important synthetic challenges in graphene chemistry that remains to be addressed is to develop a straightforward approach for the bulk synthesis of graphene functionalized with several molecules, each of them endowed with specific functions we developed a one-pot multifunctionalization of graphene with three different orthogonally-protected derivatives of 4-aminobenzylamine obtaining high degrees of addition and bulk homogeneity. More precisely, graphene was covalently functionalized through a one-pot reductive pathway using graphite intercalation compounds (GICs), in particular KC8, with three different orthogonally protected derivatives of 4-aminobenzylamine. We have employed (temperature-dependent) statistical Raman spectroscopy, X-ray photoelectron spectroscopy, magic angle spinning solid state ¹³C NMR, and a characterization tool consisting of thermogravimetric analysis coupled with gas chromatography and mass spectrometry (TG-GC-MS) to unambiguously demonstrate the covalent binding and the chemical nature of the different molecular linkers. This work may serve as a guideline for the design of 2D multifunctional materials of great interest in biomedicine, electronics, sensing, or energy storage and conversion.

A broad interdisciplinary research effort is pursued on biomedical applications of 2D materials beyond graphene, due to their unique physicochemical properties (6). We are interested to exploit these materials as drug delivery systems, highly efficient photothermal modalities, and multimodal therapeutics with non-invasive diagnostic capabilities (Figure 4). A crucial limitation of some of the 2D materials is their moderate colloidal stability in the aqueous or physiological media. The lack of suitable functionalisation strategies has encouraged the exploration of novel chemical methodologies on that front. We are exploring possible chemical modifications of 2D materials as valuable alternative to graphene.

1. Vacchi, I. A. et al. (2016) Nanoscale 8, 13714-13721.
2. Vacchi, I. A. et al. (2018) 2D Mater. 5, 035037.
3. Vacchi, I. A. et al. (2020) Chem. Eur. J. 26, 6591-6598.
4. Guo S. et al. (2020) Angew. Chem. Int. Ed. 59, 1542-1547.
5. Lucherelli M. et al. (2019) Chem. Eur. J. 25, 13218-13223.
6. Kurapati, R. et al. (2016) Adv. Mater. 28, 6052-6074.

3. Carbon nanodots

Carbon nanodots are highly promising nanomaterials for future clinical translations as they combine numerous characteristics including high photostability, low cytotoxicity, and superior biocompatibility. We have conceived and synthesised multifunctional carbon nanodots with deep-red emission properties through their controlled chemical modification using the folic acid ligand (Figure 5). These carbon nanodots, endowed of a high colloidal stability and an enhanced luminescence, are suitable for targeted intracellular production of reactive oxygen species by laser irradiation leading to efficient cancer cell death.

Our approach is radically novel and of general applicability, thus it will open new pathways towards the development of a large variety of other multifunctional carbon nanomaterials for phototherapies. In particular, the ad-hoc synthesis of nanomaterials with intrinsic therapeutic properties offers multiple opportunities for combined therapies by taking full advantage of the combination of appropriate anticancer drugs, targeting and fluorescent probes, and photothermal agents.

1. (1) Ji, DK. et al. (2020) Nanoscale Horizons 5, 1240.