Nanocarbon-based Glycoconjugates as Multivalent Inhibitors of Ebola

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Nanocarbon-based Glycoconjugates as Multivalent Inhibitors of Ebola Virus Infection Laura Rodriguez-Pérez, Javier Ramos-Soriano, Alfonso Pérez-Sánchez, Beatriz M. Illescas, Antonio Muñoz, Joanna Luczkowiak, Fatima Lasala, Javier Rojo, Rafael Delgado, and Nazario Martín J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b03847 • Publication Date (Web): 17 Jul 2018 Downloaded from http://pubs.acs.org on July 17, 2018

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Journal of the American Chemical Society

Nanocarbon-based Glycoconjugates as Multivalent Inhibitors of Ebola Virus Infection Laura Rodríguez-Pérez,‡,† Javier Ramos-Soriano,‡,† Alfonso Pérez-Sánchez,‡,† Beatriz M. Illescas,*,† Antonio Muñoz,‡ Joanna Luczkowiak,⊥ Fátima Lasala,⊥ Javier Rojo,*, § Rafael Delgado*, ⊥ and Nazario Martín*,†, #. †

Departamento de Química Orgánica, Facultad de Química, Universidad Complutense de Madrid, Av. Complutense s/n, 28040 Madrid (Spain). §

Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), CSIC – Universidad de Sevilla, Av. Américo Vespucio 49, 41092 Seville, Spain. ⊥

Laboratorio de Microbiología Molecular, Instituto de Investigación Hospital 12 de Octubre (imas12), 28041 Madrid, Spain.

#

IMDEA-Nanoscience, Campus Cantoblanco, 28049 Madrid, Spain.

ABSTRACT: SWCNTs, MWCNTs and SWCNHs have been employed as virus mimicking nanocarbon platforms for the multivalent presentation of carbohydrates in an artificial Ebola virus infection model assay. These carbon nanoforms have been chemically modified by the covalent attachment of glycodendrons and glycofullerenes using the CuAAC “click chemistry” approach. This modification dramatically increases the water solubility of these structurally different nanocarbons. Their efficiency to block DCSIGN mediated viral infection by an artificial Ebola virus has been tested in a cellular experimental assay finding that glycoconjugates based on MWCNTs functionalized with glycofullerenes are potent inhibitors of viral infection.

INTRODUCTION In the burgeoning area of nanoscience, scientists are currently focusing their attention on carbon nanomaterials for biomedical applications. The chemical modification of these nanomaterials is allowing to increase their biocompatibility and to provide new biological properties.1 Some carbon-based nanostructures have been used like drug delivery vectors,2 photothermal agents for inactivation of solid tumors,3 and photosensitizers in the singlet oxygen production, to name a few.4 Specifically, single wall carbon nanotubes (SWCNTs), multiwall carbon nanotubes (MWCNTs) and single wall carbon nanohorns (SWCNHs) are interesting as biosensors,5 scaffolds in tissue engineering,6 adjuvants for stimulating immune system7 and bioimaging.8 These carbon nanostructures have, in addition, unique properties such as electrical properties (high carrier mobility), physical properties (high surface-to-volume ratio), mechanical properties (outstanding robustness), chemical stability and versatile chemistry (covalent and non-covalent).9 Despite their appealing properties, toxicity is a handicap for the applicability of these materials.

This drawback has successfully been overcome through an appropriate chemical modification that prevents the asbestoslike behavior typically shown by these pristine carbon-based structures.10 At the molecular level, there is a wide variety of examples where multivalency drastically enhances the interactions between biomolecules in comparison with the monovalent binding. In particular, multivalency plays a key role in the proteinglycan recognition events which usually take place in the initial steps of pathogenic infection and also at some stages of the immune response.11 The search for high-affinity ligands for the study and understanding of the mechanisms involved in multivalent interactions has yielded a wide variety of artificial glycoconjugates12 which include, among others, glycopolymers,13 glycodendrimers,14 glyconanoparticles15 and glycofullerenes.16 SWCNTs and SWCNHs constitute a less-explored type of unconventional and biocompatible scaffolds for the preparation of new glycoconjugates for a multivalent presentation of carbohydrates.

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al )

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Scheme 1. Synthesis of nanoglycoconjugates. i) TBAF, NMP, 2h; ii) CuBr·SMe2, sodium ascorbate, Cu0, 60 °C, 6-7 days. DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin) is a C-type lectine expressed on dendritic cells. DC-SIGN acts as a universal pathogen receptor in the human immune system. It recognizes mannosylated and fucosylated glycans in a Ca2+ dependent multivalent way and this recognition process is the first stage for infection by some viruses, like HIV and Ebola.17,18 Therefore, one strategy to obtain antiviral agents against HIV and Ebola infections is the design of glycoconjugates which mimic the surface of the virus and interfere with the infectious process blocking the corresponding cell-surface receptor.19 In this sense, Ebola virus has a filamentous structure characteristic of a thread virus which resembles the form of SWCNTs, which usually have a diameter of 1.5-3.0 nm and a length of 20-1000 nm. MWCNTs, on their side, differ from SWCNTs in that they present a multi-walled structure, which gives them a higher rigidity. The number of concentric walls is usually in the range of 6-25, or even more, with diameters typically between 3-30 nm and a high aspect ratio that can vary from ten to ten million.9b On the other hand, HIV virus has a roughly spherical form with a diameter of about 120 nm, very similar to the form and size of SWCNHs. These

SWCNHs have a complex 3D structural modeling of Dahlia Flower shape, with multitude of concentric carbon nanocones with average diameters of 50-100 nm (similar size and shape than many common viruses). Therefore, we thought that SWCNTs, MWCNTs and SWCNHs could be employed as innovative scaffolds which, upon chemical modification, should be able to interact with DC-SIGN in a multivalent way. In the last years, glycofullerenes, fullerenes appended with different carbohydrate moieties, have focused research interests on their biological properties.16d After the first glycofullerene synthesis reported by Vasella and Diederich,20 some other monoadducts of [60]fullerene containing from 1 to 12 carbohydrate moieties have been reported.21 These derivatives, however, lack water solubility if they contain 1-2 sugar units. With more carbohydrates in their structure, they are completely soluble in water but their amphiphilic character causes their aggregation in polar media.16b, 22 Although this aggregation can be useful for some biological applications, i.e. those based in multivalent interactions,16b, 23 it also could be a drawback. Recently, Nierengarten and us have reported


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Journal of the American Chemical Society the synthesis of hexakis-adducts24 of [60]fullerene appended with 12-120 sugar copies in which the fullerene core is completely surrounded by carbohydrates and these derivatives have been evaluated for different biological applications.16d, 16h, 25 In particular, we have shown that fullerene sugar balls, namely hexakis-adducts of [60]fullerene appended with 12, 24 or 36 mannose moieties, act as strong inhibitors for DCSIGN in an Ebola infection assay model.16a, 16h, 19, 26 Furthermore, a drastic increase in the inhibition process to the subnanomolar scale has been observed when the size and number of ligands are increased in the tridecafullerenes endowed with 120 mannose units decorating the periphery of the molecule.25a In this work, we report the synthesis of new multivalent hybrid glycoconjugates based on the use of glycodendrons and suitably functionalized [60]fullerene sugar balls as appendages on SWCNTs, MWCNTs and SWCNHs as carbon based scaffolds. Characterization of these materials has been thoroughly carried out using FTIR, Raman, TGA, XPS and TEM. The antiviral efficiency of the new derivatives has been tested in an experimental infection assay using Ebola virus glycoprotein (EboGP) pseudotyped viral particles on Jurkat cells expressing DC-SIGN. This assay gives an estimate of the interaction of the glycoconjugates with DC-SIGN, since the viral entry and infection is, in this test, completely dependent on DC-SIGN.18,27 Interestingly, the new glycoconjugates do not show any cytotoxic effect at the concentration range investigated in the infection experiments.

RESULTS AND DISCUSSION Synthesis and Characterization The synthetic strategy followed for obtaining the new hybrid materials has been performed in an analogous manner for all the carbon nanoforms (Scheme 1). Briefly, utilizing the in situ generation of aryl diazonium compounds, oxidized SWCNTS (see ESI), pristine MWCNTs or pristine SWCNHs were reacted with 4-[(trimethylsilyl)ethynyl]aniline in the presence of isoamyl nitrite.28 The resulting covalent derivatives, SWCNT-TMS, MWCNT-TMS and SWCNH-TMS, were used as platforms to connect the asymmetric azidesubstituted hexakis-adducts of [60]fullerene 1-(Man) and 1(Gal) or glycodendron 2-(Man), appended with mannose (Man) or galactose (Gal) carbohydrate moieties, respectively (see ESI).25a,29 The phenylacetylene modified carbon nanoforms with exposed alkyne groups SWCNT-TMS, MWCNT-TMS and SWCNH-TMS were submitted to a deprotection step using tetrabutylammonium fluoride (NBu4F) and subsequently reacted with the corresponding azide sugar [1-(Man), 2-(Man) or 1-(Gal)] using the Cu(I)catalyzed alkyne-azide 1,3-dipolar cycloaddition reaction (CuAAC).30 Successive ultrasound bath sonication-filtration through a 0.1 µm membrane cycles yielded the functionalized glycoconjugates SWCNT-1-Man, SWCNT-2-Man, MWCNT-1-Man, MWCNT-2-Man, SWCNH-1-Man, SWCNH-2-Man and SWCNH-1-Gal. The collected solids were characterized using thermal, spectroscopic and microscopic techniques.

Figure 1. TGA under inert conditions of pristine SWCNTs (black), oxidized SWCNTs (brown), and the nanoconjugates SWCNT-1-man (red), and SWCNT-2-man (purple).

Evidences of the sidewalls functionalization was firstly obtained by thermogravimetric analysis (TGA) providing quantitative estimation of the degree of covalent functionalization on all carbon nanoforms (CNFs) (Figures S1-S3). Under nitrogen atmosphere, pristine CNFs are stable up to 800 °C. However, the oxidation process undergone on the SWCNTs produces a weight loss of 26.85 % located mostly around 300 °C which can be related to oxygenated groups anchored on the material surface mainly carboxylic acid groups. After aryl modification, the weight loss measured for the decomposition of the organic phenylacetylene groups is located between 100-600 °C. SWCNT-TMS exhibits a 5.52 % increase of the degree of surface functionalization with a total weight loss of 32.37 % (Figure 1) compared to 7.35 % for SWCNH-TMS and 9.01 % for MWCNT-TMS. This slight increase of weight loss from SWCNTs to SWCNHs and MWCNTs could be related to the different surface reactivity of the three carbon nanoforms. It is well known that carbon nanoforms curvature has an influence on their reactivity, but, even though SWCNTs and SWCNHs present a similar small diameter, the short tube length of SWCNHs (40-50 nm) and their tendency to associate into spherical structures probably leave the sidewalls less accessible for chemical reactions than in SWCNTs, thus for SWCNHs more functionalization should be observed in the tips, ended by five-pentagon conical caps.31 These reactive sites are also present along the MWCNTs structure where the side walls and edges are susceptible of chemical modification. After conjugation using click chemistry, the linkage of the glycofullerene or the glycodendron results in an important increase of the weight loss. The thermograms of SWCNT-1-Man and SWCNT-2-Man show a similar additional weight loss of 50.08 % and 45.69 % respectively at the temperature previously mentioned related to the decomposition of the asymmetric sugar balls or the dendron of mannose. For SWCNH-1-Man, the total weight loss is 30.26 % (23 % additional to SWCNH-TMS), while for SWCNH-2Man 25.54 % was observed (18 % additional to SWCNHTMS). The degree of functionalization in MWCNTs can be estimated according to TGA in a weight loss of 24.08 % for MWCNTs-1-Man and 27.73 % for MWCNT-2-Man. Additional insights of the functional nature of the CNFs were gathered using the Raman spectroscopy (Figures S4S7). The covalent binding of the phenylacetylene groups were recorded upon 785 nm laser wavelength for SWCNTs


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and MWCNTs, and 532 nm for SWCNHs. First of all, we noticed an important increase in the relative intensity of the disorder-induced mode (D-band, at 1292 cm-1) after wet oxidation of the pristine SWCNTs and an intensification of the bandwidth for the tangential mode (G-band, at 1590 cm1 ). Both band changes have been considered as probes of SWCNTs wall integrity or functionalization on the tube walls.32 The ID/IG ratio is used to calculate the amount of covalent functionalities anchored on the CNFs surface and suggests a substantial rehybridization from sp2 carbon atoms to sp3 for SWCNT-COOH. This structural modification has an important impact on the SWCNTs radial breathing mode bands (RBMs, between 186 and 270 cm-1) which are highly sensitive to the diameter and chirality and is a commonly used technique to evaluate the diameter of SWCNT revealing the presence of metallic and semiconducting SWCNTs.33 In this regard, after nitric acid treatment, we observe a disappearance of the RBMs bands. This is probably because as the SWCNTs are more defective, they are more susceptible to bundle to each other, and the bundling effect caused the vibrational damping.34 After the aryl diazonium chemistry reaction only changes in the D band are observed for SWCNT-TMS with a ID/IG ratio growth related with the anchoring of phenylacetylene groups. However, this ID/IG value remains almost constant for SWCNT-1-Man and SWCNT-2-Man, proving that the SWCNTs framework is unaffected under the reaction conditions used in the CuAAC chemistry.

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bonaceous material follows the same behavior that was found for SWCNTs and SWCNHs with a small increase of the ID/IG ratio from pristine MWCNTs to MWCNT-TMS followed by stagnation after click chemistry to form MWCNT-1-Man and MWCNT-2-Man. The infrared spectroscopy (FTIR) has been a useful tool for the identification of known functional groups present in the intermediates and final conjugates (Figures S8-S13). In this regard, the band corresponding to the acetylenic spacer, at 2159 cm-1, related with the protected alkyne groups anchored in SWCNT-TMS, MWCNT-TMS and SWCNHTMS, disappears after the CuAAC reaction due to the formation of the triazole ring. Furthermore, the carbonyl vibration due to the ester units of the fullerene appended conjugates is seen at around 1740 cm-1 for the three CNFs derivatives. The absence of the azide groups (2092 cm-1) present in the asymmetric glycofullerenes and dendron highlights that no unbounded residues remain in the final products (Figure 3).

SWCNH-p SWCNH-TMS SWCNH-2-Man

a.u. 0.99

ν (C=C) 1575 cm-1 ν (C-H) stretching

0.96

ν (C≡ C) 2159 cm-1

0.93

ν (C=O) 1736 cm-1 -1

ν (C-O) 1116 cm stretching 0.90 4000

3000

2000

1000

Wavelength (cm-1)

Figure 3. FTIR characterization of pristine SWCNH (black), SWCNH-TMS (blue) and the final covalent material SWCNH2-Man (pink).

Figure 2. Raman spectra of pristine MWCNT (black), MWCNT-TMS (blue), MWCNT-1-Man (red) and MWCNT2-Man (pink) under 785 nm laser excitation wavelength.

The Raman spectra of pristine SWCNHs and MWCNTs have two bands with approximately equal scattering strengths detected around 1591 and 1346 cm−1 attributed to the “G band” and “D band”, respectively33,35 (Figure 2). The D band in the SWCNHs is attributed to the loss of the basal plane lattice due to the ending by a five-pentagon conical cap and to the sp3 single-bonding carbon atoms existing within SWCNHs aggregates.36 Therefore, the SWCNH-TMS Raman spectra reveal a discrete enhancement in intensity for this band derived from the grafting of the aryl groups, remaining nearly unaltered for SWCNH-1-Man and SWCNH-1-Gal. Raman spectra of MWCNTs presents a D band related with structural defects on their walls. This car-

To further corroborate the nanomaterial characterization presented so far, we recorded the X-ray photoelectron spectroscopy (XPS) to analyze the elemental composition of the CNFs surface. The survey spectra recorded for all new conjugates display the electrons collected from C 1s, O 1s, N 1s and Si 2p core levels. Other elements, such as, Cl, Al,…, which are attributed to solvent or surface impurities of the deposition method, are also present in very low concentration (< 1%) and were not considered for atomic concentration (%) calculation. It should be noticed the absence of N 1s in the spectra registered for the intermediate materials MWCNT-TMS, SWCNT-TMS and SWCNH-TMS (Figures 4 and S14-S17) together with the presence of a Si 2p signal from the protecting group after the reaction with the in situ generated aryl diazonium compound (Tour reaction). The emission features of oxygen is present in all the samples, from the pristine material to the final conjugates, which is likely to originate from the atmospheric gases absorption due to the high surface area and microporosity, especially in the case of SWCNHs.37 However, the oxygen content increases drastically upon glycofullerene functionalization, as expected. As an example, the semiquantitative analysis of


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Journal of the American Chemical Society MWCNT-1-Man and MWCNT-2-Man indicates an oxygen content of 9.3% and 14.6% respectively compared to the 4.8% measured for MWCNT-TMS. The same behavior is observed for the SWCNTs and SWCNHs derivatives. The high resolution spectra were then recorded for the C 1s, O 1s and N 1s. All peaks were decomposed into several symmetrical (Gaussian-Lorentzian curves) components. The C1s peak of all the CNFs was adequately fitted to five components (sp2 C=C, sp3 C-C, C–O, C=O and COO groups) according to the peak assignment reported in the literature.38 The N 1s core signals observable for SWCNT-1-Man, SWCNT-2-Man, MWCNT-1-Man, MWCNT-2-Man, SWCNH-1-Man, SWCNH-1-Gal and SWCNH-2-Man are centered around 400 eV (Figures S14-S17). Analyzing the N 1s fitting, we observe the same behavior for all the final sugar carbon nanoform materials, one major peak at lower binding energies assigned to two N-atoms bound to C-atoms as near neighbors and another peak belonging to the third nitrogen of the triazole ring.39 Note that no peak at 405.0 eV assigned to the azido group is observable, which confirms the lack of free unbounded organic molecule physisorbed on the material surface and, therefore, indicates their covalent attachment to the CNFs, as previously confirmed by FTIR results.40

However, it is important to highlight the enormous tendency of SWCNTs to originate large bundles due to attractive interactions between them such as π-π staking and London forces even after oxidation and covalent functionalization (Figure S19). On the other hand, MWCNT have the same morphology as the SWCNTs with no aggregation limitations, leaving all the active functional mannose groups free to be evaluated in the biological activity study. The structure of SWCNHs and their “Dahlia like” spherical aggregation are highly preserved after the chemical modification process (Figures S23 and S24) Furthermore, the hydrodynamic radius average found for those final materials estimated by dynamic light scattering (DLS) in water is 195 nm for both MWCNTs-1-Man and MWCNT-2-Man. A larger size distribution is found for SWCNTs, where two main contributions are observed with values of 116 and 492 nm for SWCNT-2-Man (Figures S25S27). The spherical aggregation of SWCNH-1-Man and SWCNH-2-Man provides good water solubility under ambient conditions, with values of 238 nm and 214 nm respectively, being very similar to the one measured by TEM.

Figure 5. Representative TEM micrographs SWCNH-1-Man. Insets show the tip of SWCNH-1-Man to which C60-like molecules are attached. Figure 4. SWCNH-1-Man (red) and SWCNH-1-Gal (green) XPS spectra compared to SWCNH-TMS (blue). XPS N 1s deconvolution for both SWCNH-1-Man (top left) and SWCNH-1-Gal (top right).

Studying the morphology of the new glycoconjugates by transmission electron spectroscopy (TEM) is important to understand the behavior of the carbon nanoforms in the biological assays. For SWCNT-1-Man, MWCNT-1-Man and SWCNH-1-Man we have observed small C60 like round shaped molecules attached to the CNTs side wall surface or the SWCNHs tips (Figure 5). The diameter measured for these spherical objects was around 1 nm (see width profile in Figures S18-S24), being the expected for the corresponding [60]fullerene derivative. For SWCNT-2-Man, MWCNT-2Man and SWCNH-2-Man, TEM micrographs shows the different CNFs with densely covered walls compared with the pristine materials, which can be related with the organic dendron functionalization.38

Biological Studies The antiviral activity of nanoglycocomposites decorated with mannose moieties SWCNT-1-Man, SWCNT-2-Man, MWCNT-1-Man, MWCNT-2-Man, SWCNH-1-Man and SWCNH-2-Man was tested using pseudotyped viral particles presenting at the surface Ebola virus Glycoproteins (GP) which bear the pNL4−3Luc plasmid that encodes the luciferase biosynthesis and acts as a reporter of the infection. The infected cells will show activity, quantifiable with a spectrophotometer, in presence of luciferine (Figure S29). We have applied an assay based on GP-pseudotyped lentiviral particles that has been extensively used for pathogenesis and antiviral screening by our group and others.29, 41 The usage of the pseudotype systems for viral neutralizatio,n or antiviral screening has been compared to live viruses showing a strong correlation with many different viruses such as: HIV1, Ebola virus, SARS, Coranovirus, Nipah virus, Influenza virus, Rabies virus etc.42 The inhibition of cis-infection process of DC-SIGN+ cells was evaluated in the presence of carbohydrate-based conjugates SWCNT-1-Man, SWCNT2-Man, MWCNT-1-Man, MWCNT-2-Man, SWCNH-1-



Man and SWCNH-2-Man at different final concentrations. The values described in Table 1 correspond to three independent experiments. The results of blocking DC-SIGN receptor by different compounlds were shown as a function of concentration. The 50% of inhibition of the infection (IC50) was calculated with the 95% confidence intervals (Table 1). As a control, infection with VSV-G pseudotyped retroviruses, which is independent of the presence of DC-SIGN receptor, was performed under the same conditions. The results obtained in the cis-infection experiment revealed the dependence of the inhibition effect on mannoses. The nanoglycocomposite SWCNH-1-Gal displaying galactoses moieties was used as negative controls and, as expected, were not able to inhibit the infection process mediated by DC-SIGN. The glycosylated carbon nanoforms had a different performance facing DC-SIGN. Both SWCNTs (SWCNT-1Man and SWCNT-2-Man), with their rod-like shape, show antiviral activity, blocking DC-SIGN receptor with a IC50 of 15.15 and 2.20 µg/mL, respectively (Table 1). In contrast, MWCNTs conjugated with glycofullerenes (MWCNT-1Man) block the receptor in a more efficient manner than MWCNT-2-Man, with IC50 values of 0.37 and 1.90 µg/mL, respectively. However, the antiviral activity of MWCNT-2Man is similar to the SWCNT analogous (SWCNT-2-Man). On the other hand, SWCNHs conjugated with glycofullerenes (SWCNH-1-Man), with their globular structure, are able to inhibit Ebola Virus infection more efficiently than glycodendron derivatives (SWCNH-2-Man), blocking DC-SIGN receptor with a IC50 of 33 and 202 µg/mL, respectively (Table 1). To find an explanation to these results, we have carried out spectrophotometric measurements of the sugar content in each type of glycoconjugate following the anthrone method.43 For each type of CNF, the most active derivative is that with more mannose content. On the other hand, the low activity of SWCNHs in comparison with SWCNTs and MWCNTs must be related, not only with the lower presence of mannose in these carbon nanoforms, but also with the size and shape of these carbon nanoforms (Table 1). Comparing these results with the previously obtained for the hexakis-adduct of [60]fullerene substituted with 12 mannose units C60-12Man,26 we observe an increase in the antiviral activity in the case of glycoconjugates based on SWCNTs and MWCNTs, while mannose derivatives of SWCNHs are less active than the glycofullerene itself. MWCNTs possess an ideal natural structure for this purpose, and attaching a proper glycosylated fragment, they can act as mimetics of a highly glycosylated viral capsid. On the other hand, the glycodendrimer 2-(Man) does not show any antiviral activity DC-SIGN mediated at a concentration of 5oo µg/mL. This finding demonstrates the importance of the multivalent presentation of the carbohydrate ligands provided by the CNF scaffold. In previous studies carried out with other glycodendrons in the same infection model, it was found that at least 24 copies of the ligand were needed to have a potent inhibition activity.27, 44 The cytotoxic effect of all CNF glycoconjugates was studied by a cell proliferation assay using the Cell Titer 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega). Notably, there was not any appreciable cytotoxic

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effect of these materials at the concentration range investigated in the infection experiments (Figure S30). Table 1. IC50 values obtained for new glycoconjugates and mannose quantification.

Glycoconjugate

IC50 (μg/mL)

Quantity of Man (μg/mg)a

-

426

19.01 15.15 2.20 0.37 1.90 33 202

226 30.5 32.5 26.4 21.8 13.9 10.6

2-(Man) C60-12Man SWCNT-1-Man SWCNT-2-Man MWCNT-1-Man MWCNT-2-Man SWCNH-1-Man SWCNH-2-Man

SUMMARY AND CONCLUSIONS In summary, we have carried out the synthesis of new carbon-based nanohybrids in which different sized and shaped SWCNTs, MWCNT and SWCNHs have been used as scaffolds, which have been covalently connected to glycodendrons with 9 mannose units and glycofullerenes constituted by 10 monosaccharides of different nature (mannose or galactose). After a Tour reaction to link the aryl group with alkyne moieties, the well-known CuAAC “click” reaction has been successfully used to bind the glycodendrons and glycofullerenes to all carbon nanoforms. A slightly higher degree of functionalization results for SWCNTs and MWCNTs when compared with SWCNHs, which can be justified by the higher surface available for chemical reaction in the latter cases. All new nanohybrids have been fully characterized by different experimental techniques (FTIR, Raman, TGA, XPS and TEM), which nicely confirm the proposed chemical structures. Interestingly, TEM micrographs show the presence of spherical C60 units covalently connected to the carbon nanotubes walls. Most importantly, the new carbon-based nanohybrids have been tested as inhibitors of Ebola virus infection. In this regard, MWCNT-1-Man, in which the MWCNTs are connected to the glycofullerenes, presented the higher biological activity. In contrast, SWCNH-2-Man exhibited the lowest activity. Considering each type of carbon nanoform, the biological activity can be related with the amount of sugar content. An increase of the RIP, the relative inhibitory potency, can be calculated for SWCNTs and MWCNTs derivatives. Consequently, significant interaction with the DCSIGN receptor and inhibition of the virus infection at concentrations as remarkable as 0.37 µg/mL were observed. These experimental findings reveal that MWCNTs are efficient carbon-based scaffolds whose bio-compatibility and size make them very appealing materials to be used in a broader sense as efficient carbon-based platforms for biomedical applications.26

AUTHOR INFORMATION Supporting Information Synthetic procedures and characterization details. Supporting figures. Biological assays description.


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Journal of the American Chemical Society Corresponding Author * [email protected] * [email protected] * [email protected] * [email protected]

Author Contributions ‡ These authors contributed equally.

ACKNOWLEDGEMENT Financial support by the European Research Council (ERC320441-Chirallcarbon), Ministerio de Economía y Competitividad (MINECO) of Spain (projects CTQ2017-83531-R, CTQ2017-84327-P and CTQ2014-52328-P), the Comunidad Autónoma de Madrid (PHOTOCARBON project S2013/MIT2841), Instituto de Salud Carlos III (ISCIII) (FIS1400708 and Red de Investigación en SIDA, RIS, RD12/0017).

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