Graphene Quantum Dots Based Systems As HIV Inhibitors

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Graphene Quantum Dots based systems as HIV Inhibitors Daniela Iannazzo, Alessandro Pistone, Stefania Ferro, Laura De Luca, Annamaria Monforte, Roberto Romeo, Maria Rosa Buemi, and Christophe Pannecouque Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00448 • Publication Date (Web): 14 Aug 2018 Downloaded from http://pubs.acs.org on August 15, 2018

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Bioconjugate Chemistry

Graphene Quantum Dots based systems as HIV Inhibitors Daniela Iannazzo,*,†Alessandro Pistone,† Stefania Ferro,‡ Laura De Luca,‡ Anna Maria Monforte,‡ Roberto Romeo,‡ Maria Rosa Buemi ‡ and Christophe Pannecouque§ †

Department of Engineering, University of Messina, Contrada Di Dio, I-98166 Messina, Italy

‡

Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of

Messina, Viale Annunziata, I-98168 Messina, Italy §

KU Leuven, Department of Microbiology and Immunology, Laboratory of Virology and

Chemotherapy, Rega Institute for Medical Research, Herestraat 49, B-3000 Leuven, Belgium *

Corresponding author.

E-mail address: [email protected]; Tel: +39 090 3977569; Fax: +39 090 3977494

ABSTRACT: Graphene quantum dots (GQD) are the next generation of nanomaterials with great potential in drug delivery and target-specific HIV inhibition. In this study we investigated the antiviral activity of graphene based nanomaterials by using water soluble GQD synthesized from multi-walled carbon nanotubes (MWCNT) through prolonged acidic oxidation and exfoliation and compared their anti-HIV activity with that exerted by reverse transcriptase inhibitors (RTI) conjugated with the same nanomaterial. The antiretroviral agents chosen in this study, CHI499 and CDF119, belong to the class of non-nucleoside reverse transcriptase inhibitors (NNRTI). From this study emerged the RTI-conjugated compound GQD-CHI499 as a good potential candidate for HIV treatment, showing an IC50 of 0.09 µg/mL and an EC50 value in cell of 0.066 µg/mL. The target of action in the replicative cycle of HIV of

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the drug conjugated samples GQD-CHI499 and GQD-CDF119 was also investigated by a time of addition (TOA) method, showing for both conjugated samples a mechanism of action similar to that exerted by NNRTI drugs.

KEYWORDS: Graphene quantum dots, antiretroviral agents, HIV therapy

BRIEFS: Graphene quantum dots (GQD) conjugated with reverse transcriptase inhibitors (RTI) showed to be potential candidates for HIV treatment, worthwhile to be further investigated.

INTRODUCTION The applications of nanoscience in biotechnology, pharmaceuticals and medicine have shown the potential to revolutionize the future of medical care by developing innovative nanostructured materials for preventive, diagnostic and therapeutic purposes.1,2 The rapidly developing field of nanotechnology has produced a huge battery of nanoparticles including polymers, proteins, micelles, liposomes, dendrimers, nanocrystals, nanoshells, paramagnetic nanoparticles, gold nanoparticles and carbon nanomaterials, thus allowing the design of tailored and biocompatible nano-drugs with well defined size and shape, able to load high and fixed drug content and to release the therapeutic agent to damaged cells or tissues in a selective and controlled manner.3,4 The outstanding properties of these “smart drugs” give hope to cure or improve the health conditions of millions of people worldwide, affected with deadly diseases as cancer and infectious diseases caused by viruses such as the human immunodeficiency virus (HIV), Ebola virus (EBOV), dengue virus (DENV) and the severe acute respiratory syndrome coronavirus (SARS-CoV).


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Despite decades of intensive research towards effective and safe pharmaceutical products, the acquired immune deficiency syndrome (AIDS), a disease caused by HIV, still represents a major public health crisis worldwide, affecting approximately 36.9 million humans.5 Presently, there is no preventive vaccine for AIDS and the HIV prophylaxis (pre- and post-exposure prophylaxis) is mainly based on the use of combinations of antiretroviral drugs.6 The use of the highly active antiretroviral therapy (HAART), which uses a combination of drugs targeting different steps of HIV life cycle, has significantly improved the life expectancy and life quality of patients affected by AIDS. However, this combination therapy must be taken for a lifetime in order to prevent re-emergence of HIV from latently infected cells; it gives serious adverse effects and is ineffective where the virus develops resistance, often leading to clinical failure in the treatment.7 Moreover, the efficacy of many effective therapeutic agents is limited by their low solubility in the solvent used for their administration and their cytotoxic side effects.8 Therefore, there is an urgent need to develop alternative anti-HIV approaches, able to radically advance the treatment and prevention of AIDS. Among the different classes of nanomaterials, the carbon allotropes fullerenes, carbon nanotubes and graphene, due to their transporting abilities combined with the appropriate surface modifications and their fascinating chemical, physical and biological properties, have attracted particular attention in the development of nanocarriers for anticancer drugs delivery and nano-drugs for anti-HIV therapy.9-11 Some of these nanomaterials have therapeutic effects by themselves, with efficacy and biocompatibility strongly depending from their physical and chemical properties. Cationic C60 bis-fulleropyrrolidines, bearing ammonium groups, showed strong antiviral activity on the replication of HIV-1 in human CD4+ T cells;12 C60 fullerene derivatives bearing several carboxyl groups, even if less active against HIV, showed enhanced solubility in water and low toxicity.13 Graphene and graphene oxide (GO) have also been investigated as HIV inhibitors.14,15 Carbon nanotubes have been investigated as anti-HIV agents by experimental16,17 and computational studies.18 Differently functionalized multi-walled carbon nanotubes (MWCNT) have also shown good in vitro anti-HIV activity, which was proved to be strictly


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related to the hydrophilicity and dispersibility of the nanosystems.17 Graphene quantum dots (GQD), small fragments of monolayer graphene sheets, represent s the next generation of carbon-based nanomaterials with tremendous potential in a broad range of biomedical applications.19,20 These nanomaterials have demonstrated to be more hydrophobic and less toxic with respect to graphene and are endowed with stable strong fluorescence, thus allowing the human cell tracking.21-24 Moreover, similarly to other graphene based nanomaterials, the presence of several functional groups on their surface allows their multimodal conjugation, making them ideal materials for drug delivery and target-specific HIV inhibition.25,26 Even if the study of graphene-based antivirals is still at a nascent stage, graphene quantum dots, due to their tunable photoluminescence, colloidal stability and biocompatibility have demonstrated the potential to revolutionize the future of anti-HIV therapy. GQD conjugated with boronic acid moieties have shown the ability to prevent HIV-1 infection via interaction with gp120, thus avoiding the target cell interaction.26 In this study, we have investigated for the first time, the antiviral activity of GQD, synthesized by prolonged acidic oxidation and exfoliation of MWCNT, as inhibitors of the reverse transcriptase of HIV which represents the main target for the clinical treatment of HIV infections. Their anti-HIV activity was compared with that exerted by known inhibitors of reverse transcriptase which were conjugated with the same nanomaterial (Figure 1). The antiretroviral agents chosen in this study, CHI499 and CDF119, belong to a series of active NNRTI.27,28 The conjugation of these poorly water soluble drugs with GQD was proved to afford to high water dispersible systems, thus opening new opportunities in the use of other clinically used poorly water soluble anti-HIV drugs endowed of critical cytotoxic side effects. The obtained results clearly point out the physicochemical properties of GQD-based nanomaterials as important features for the antiviral activity and highlight the anti HIV activity of GQD and the improved anti-RT activity of the NNRTI-conjugated compounds.


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Figure 1. GQD and GQD-RTI conjugates.

RESULTS AND DISCUSSION Synthesis and characterization of GQD The water soluble GQD used for this study were prepared by prolonged acidic oxidation of pristine MWCNT following a previously reported procedure.29 The presence of many defects in the graphene sheets of MWCNT, greatly contributes to the formation of nanodots with many oxygen-containing functional groups and improved water solubility.30 The obtained material was characterized by XRD, DLS, PL and Raman spectroscopy and their morphology was investigated by HRTEM (Figure 2). As expected, the XRD spectrum of GQD sample shows no diffraction signals in the entire 2 theta range; in particular, the (002) XRD peak that usually represents a measure of interplanar spacing for two neighbouring graphene layers,31 is present in the spectrum of the precursor MWCNT and is totally


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absent in the newly synthesized nanomaterials, thus confirming the single layer structure after acidic exfoliation (Figure S1 ESI). The morphologies and structure of GQD were investigated by transmission electron microscopy (TEM) and by atomic force microscopy (AFM). The representative TEM images of the synthesized nanodots show monodisperse nanoparticles with circular shape and uniform diameter less than 5 nm in size (Fig. 2A); moreover, the high resolution TEM image indicates a crystalline graphene structure (inset in Fig. 2A). The presence of a graphene structure which allows the easy surface functionalization with therapeutic agents together with the uniform and small particle size makes GQD as ideal nanopharmaceuticals, able to cross biological barriers and to access into damaged cells.32 The AFM image of GQD, reported in Figure S2, shows GQD with diameters distributed mainly in the range of 5-10 nm with round shape. The particle size measured by DLS (Fig. 2B) showed a smaller size population with an hydrodynamic radius centered at 12.2 nm (88%) and a larger population with an average hydrodynamic radius of 44.8 nm (12%). The width of the observed distributions are approximately Gaussians, thus indicating the sample polydispersity. The different size values observed by TEM and DLS analyses can be explained by the possible aggregation of GQD in water dispersion where non-covalent interactions and hydrogen bonding electrostatic attraction between the oxygen functionalities can occur. Moreover, the relatively broad particle distribution indicates the various orientations of these nanoparticles toward the laser and detector used for the measurements.33 The optical properties of GQDs were investigated studied using UV−vis and PL spectra (Figures S3 and S4, ESI). The UV−vis spectrum of GQD sample (Figure S3) shows the absorption band at ~250 nm corresponding to the π (bonding) –π* (antibonding) transition of sp2 aromatic domains. The homogeneous distribution of the nanoparticle size was also confirmed by PL spectra (Figure S4, ESI). Even exciting the dispersions of GQD at the wavelengths in the range of 330-370 nm, always a strong peak at 560 nm was observed. The Raman spectra of GQD and of the precursors pristine MWCNT show the presence of the D (1320 cm-1) and G (1594 cm-1) peaks, representative of the aromatic domains. The D peak of GQD is more intense than the G one, when compared to the MWCNT spectrum, thus further


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confirming the loss of long range order after the strong oxidative reaction (Figure S3, ESI). The prolonged acidic treatment and long-time sonication have proved to exert a positive effect in increasing the amount of acidic groups on the graphene surface. In fact, the titration analysis performed on the synthesized GQD as a function of their zeta potential, showed an increased amount of acidic group (2.37 mmol/g) with respect to what observed after only 6 hours of treatment for a previously reported oxidized MWCNT sample (1.8 mmol/g).17 Moreover, this strong oxidation treatment greatly enhances the dispersion stability of the synthesized GQD in water. This improved hydrophilicity renders these nanomaterials less reactive in the biological systems and easier to be transported along the physiological milieu, thus also accelerating their elimination.34 It is widely known that nanoparticles with zeta potentials more positive than +30 mV or more negative than -30 mV will tend to repel each other and have no tendency to aggregate.35 Thus, in order to investigate the GQD stability in water, we have calculated the zeta potential of the synthesized nanodots at different pH values. As expected, the nanoparticles will tend to acquire a more negative charge by increasing the pH of the medium but their zeta potential values are always low than -30mV in all the investigated pH range of 4-9, thus demonstrating their high stability in water (Figure 2C). Taken all together, these results prompted us to investigate the newly synthesized GQD as nanoplatforms for the treatment of HIV. .


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Figure 2. (A) Representative HRTEM images of GQD sample; (B) Intensity of size distribution of GQD sample dispersed in deionized water at concentration of 0.1 mg/mL; (C) Isoelectric titration graph of GQD sample. The pH of medium dependence of zeta potential was evaluated at concentration of GQD of 0.1 mg/mL.

Synthesis and characterization of GQD-CHI499 and GQD-CDF119 In order to investigate the combined effects of GQD hydrophilicity and the anti-HIV activity of antiretroviral drugs, the synthesized nanomaterial was conjugated with the non-nucleoside reverse transcriptase inhibitors, CHI499 and CDF119, chosen for this study as model drugs. The antiretroviral drug CDF119 was prepared following a procedure previously described by us:36


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The reduction of the nitro group of the obtained 2-{[1-(3,5-dimethylbenzyl)-1H-benzimidazol-2yl]sulfanyl}-N-2-nitrophenylacetamide was performed by treatment with stannous chloride at reflux for 2h (Scheme S1, ESI). Subsequently, the obtained residue was diluted with 10N NaOH aqueous solution and stirred at room temperature for 1h to provide, after purification, the desired compound. The anchorage of the antiretroviral drugs to the GQD surface was performed by coupling reactions between the nucleophilic amino groups of the drugs and the carboxylic functionalities present on the tubes (Scheme 1). The coupling of the carboxylic groups present on the graphene surface and the sulfonamide moiety of CHI499 was achieved using EDC and 1 equivalent of DMAP to afford the nanosystem GQDC499, while the synthesis of GQD-CDF119 was performed by coupling reaction with between the carboxylic groups of GQD, activated by the reagents EDC/HOBt, and the amino functionality of CDF199, using 0.1 equivalent of DMAP. The amount of drugs loaded on the GQD, calculated as difference between the amount of drug added to the reaction mixture and the amount of drug collected in the solutions filtered by dialysis bags and also evaluated by TGA analyses were found to be of 14.5 and 20% for GQD-CDF119 and GQD-CHI499 samples, respectively. From a chemical point of view, the electronic nature of the chemical groups nearby the newly formed C(O)-NH bond could deeply affect the drug release in biological medium. The imide bond present in the GQD-CHI499 system is easier cleavable than the amide bond present in the GQD-CDF119 system because of the favoured displacement of the sulfonamido leaving group, which allows the release of CHI499 in HIV infected cells in an easier manner with respect to the release of CDF119 from GQDCDF119.


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Scheme 1. Synthesis of GQD-CHI499 and GQD-CDF119. Reagents and conditions: (a) CHI499, EDC⋅HCl, DMAP, CH2Cl2, r. t., 4 d; (b) CDF119, EDC⋅HCl, HOBt, DMAP, CH2Cl2, r. t., 4 d.

The effectiveness of reaction coupling and the amount of drug anchored to the GQD were evaluated by TGA and by FTIR spectroscopy (Figure 3). The FTIR spectra of GQD sample show two strong peaks at 3450 cm-1 and 1620 cm-1 that are ascribable to the vibrations of O-H and C=O bonds, respectively; moreover a peak at 1072 cm-1, related to the C-O alkoxy group can be observed. (Figure 3A). These data clearly indicated the presence of many oxygenated functional groups on the GQD surface, thus justifying their high solubility in water. The FTIR spectra of the conjugate samples GQDCHI499 and GQD-CDF119 show the additional representative peaks at 1640 cm-1 and 1630 cm-1 respectively, due to the C=O stretch of the amide functionalities present on the nanosystems sample


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after the conjugation with the RTI. The spectrum of GQD-CHI499 shows the additional peak at 1310 cm-1 due to the stretching of S=O bond. Moreover, for both samples a broad peak at 2900-2950 cm-1 due to the N-H stretching of secondary amine can be observed. The TGA spectra of GQD-CHI499 and GQD-CDF119 and of their precursor GQD, performed under argon atmosphere, showed the increase of weight losses for both samples after the drugs conjugation which are directly related to the increase of organic moiety present on the GQD surface (Figure 3B). These additional weight losses, calculated at 500 °C are of 20% for GQD-CHI499 and 14% for GQD-CDF119. Moreover, different shapes of TGA profiles can be observed for the conjugated samples, thus further confirming the deep chemical changes occurred on the nanomaterials.

Figure 3. (A) FTIR spectra of GQD, GQD-CHI499, and GQD-CDF119; (B) TGA curves for GQD, GQD-CHI499 and GQD-CDF119. All the experiments were performed under argon atmosphere. In Table 1 are reported the percentages of mass losses (∆m% absolute) of the samples GQD, GQDCHI499 and GQD-CDF119 and the amount of drugs CHI499 and CDF119 conjugated to the graphene surface as percentages of weight losses calculated by difference from the precursors GQD (∆m% relative) at 500 °C under inert atmosphere; the relative calculated concentrations of antiretroviral


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compounds anchored on the GQD are also reported.

Table 1. Comparative analysis of the functionalization degree of nanosystems GQD-CHI499, and GQD-CDF119 as percentage of mass losses, calculated at 500 °C under argon atmosphere.

∆m (%) absolute

∆m (%) relative

GQD

5

-

Drug amounta (µmol/mL) -

GQD- CDF119

19.5

14.5

0.348

GQD- CHI499

25

20

0.388

Sample code

a

Calculated as mg/MW of drug in 1mg of sample

The presence of stable GQD/drugs interaction was also confirmed by PL spectra. The photoluminescence properties of GQD as well as the PL spectra of the conjugated samples at the excitation wavelength of 370 nm in deionized water, are shown in Figure 4. The PL λmax of GQD, GQD-CHI499 and GQD-CDF119 samples were about 590, 570 and 550 nm, respectively. As reported for similar studies on the covalent conjugation of GQD with small molecules, these blue-shifts of emission can prove that GQD are covalently bound to the RTI drugs.37


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Figure 4. PL spectra of GQD, GQD-CHI499, and GQD-CDF119. In order to further study the effect of the graphene surface functionalization on the dispersibility of the nanomaterials in water, we have sonicated the drug-conjugated samples GQD-CHI499 and GQDCDF119 in pure water and in phosphate buffered saline solution (PBS, pH 7.4) by dispersing 2 mg of each sample in 10 mL of solvent; as shown in Figures 5A and 5C a good dispersion stability for all the investigated samples after 2 days, can be observed. As expected, the high content of oxygen functional groups present in the GQD made this nanomaterial dispersible in water for months. These results highlight the positive effect of the presence of carboxylic functionalities on the water dispersion stability of nanomaterials and the effective approach of using nano-sized carriers as delivery systems for poorly water soluble drugs. We have also investigated the dispersibility of GQD and GQD based samples both in pure water and in PBS by calculating their electrophoretic mobility in these solvents. The DLS analyses of the investigated samples showed zeta potential values ranging from -43.9 to -42.1 for the GQD based samples dispersed in pure water and values ranging from -43.6 to -41.9 for the same samples, dispersed in PBS ((Figures 5B and 5D). The obtained zeta potential values, which were always lower than -30 mV, further confirmed the high stability of the synthesized nanosystems. in water based solvents. ACS Paragon Plus Environment

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Figure 5. Dispersibility of GQD, GQD-CHI499 and GQD-CDF119 in pure water (A) and PBS (C) and calculation of the corresponding zeta potential values in pure water (B) and PBS (D).

Anti-HIV activity of GQD, GQD-CHI499 and GQD-CDF119 The ability of all the synthesized nanomaterials to act as reverse transcriptase (RT) inhibitors of HIV was evaluated in cellular and enzyme assays and their cytotoxicity was investigated in MT-4 cell. The results of biological assays were compared with that exerted by the antiretroviral compounds CHI499 and CDF119 and by the GQD sample alone. As reported in Table 2, the GQD sample shows anti-HIV activity with an IC50 of 37.6 µg/mL, an EC50 value in cell of 19.9 µg/mL and a selectivity index of 3. A similar behaviour is observed for the conjugated sample GQD-CDF119 that maintains the selectivity index of the drug CDF119. The GQD-CHI499 instead, revealed to be a potential good candidate for


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HIV treatment; With respect to the drug CHI499 alone, the conjugate system shows a marked improvement in anti-RT and cellular anti-HIV-1 activities with a IC50 value of of 0.09 µg/mL and an EC50 value of 0.066 µg/mL (SI=362). The results obtained from biological tests allow to speculate on the mechanism of action of GQD and GQD-based materials. The polycarboxylated GQD could exert the inhibition of RT activity by inhibition of virus binding to the cells as reported in literature with similar polyanionic compounds.38 The results of enzymatic RT and cellular anti-HIV virus assays, for GQD and GQD-CDF119 showed comparable results thus suggesting as a similar mechanism of action for both materials possessing a polyanionic character. The more pronounced anti-HIV activity of GQD-CHI499, both in the RT assay and in the cellular assay, as compared to the free drug CHI499, could be explained considering the chemical nature of imide bond present in the GQD-CHI499 system. In this case, the NNRTI is easily released from the conjugate resulting in a mixture of GQD and free CHI499 in the infected cell and thus leading to a dual mechanism of action. The amide bond present in the GQD-CDF119 is more stable and allows the drug release in more harsh reaction conditions.

Table 2. Anti-RT and cellular anti-HIV-1 activities and cytotoxicity in MT-4 cells of GQD, GQDCHI499 and GQD-CDF119 and of the free drugs CHI499 and CDF119. Sample code

IC50a µg/mL

EC50b µg/mL

CC50c µg/mL

SId

CHI499

0.67 ±0.00

0.12 ± 0.05

62.82 ± 15.29

540

CDF119

4.05 ± 0.33

0.64 ± 0.22

2.71 ±0.59

4

GQD

37.6 ± 6.23

>19.90

51.5 ± 8.9

≤3

GQD-CHI499

0.09 ± 0.12

0.066 ± 0.011

23.9 ± 9.6

362

GQD-CDF119

43.3 ± 17.0

>13.2

55.8 ± 10.4

≤4

a

IC50 = concentration able to inhibit by 50% the activity of RNA-dependent DNA polymerase in vitro EC50 = effective concentration able to reduce by 50% the HIV-1-induced cytopathic effect in MT-4 cells. b


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Bioconjugate Chemistry c

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d

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CC50 = cytotoxic concentration able to reduce by 50% the MT-4 cell viability. SI = selectivity index (ratio CC50/EC50). The results obtained from the in vitro anti-HIV and RT assays as well as the evaluation of cytotoxicity

in MT-4 cells allow to confirm the hypothesis that hydrophilicity and water dispersibility are pivotal issues in determining good biological profiles in graphene based antivirals. In fact, the values obtained performing the same biological tests with the less water dispersible MWCNT, conjugated with reverse transcriptase inhibitors,17 showed an IC50 value of the more active RTI-conjugated sample equal to 4.56 µg/mL, with a low pharmacological profile (EC50 and CC50 values of >74.4 µg/mL and 94.73 µg/mL, respectively). In general, the selectivity indexes reported for GQD and GQD conjugated systems are comparable with that exerted by the free drugs, thus evidencing no major limitations for all the synthesized nanomaterials. Knowing the mechanism of action of newly synthesized antiviral agents represents an essential requisite for their clinical development. Thus, we further investigated the target of action in the replicative cycle of HIV of the drug conjugated samples GQD-CHI499 and GQD-CDF119 by a time of addition (TOA) experiment which allows to reveal the mechanism of action of anti-HIV drugs in cell culture by comparing their time of intervention with that of well-characterized inhibitors.39 In principle, the HIV replication could be inhibited if the investigated compounds were added before their time of intervention in the virus replication cycle, as measured by the production of p24 antigen, thus allowing to investigate the presumed target of new anti-HIV agents.38 The antiviral activities of the GQD conjugated samples were compared with that exerted by the NNRTI CHI499 CDF119, efavirenz (DMP266), a clinically used NNRTI and by the virus-cell-binding inhibitor dextran sulfate 8000 (DS average MW 8000; DS8000). The results of this test, shown in Figure 6, suggest a similar mechanism of action for both conjugated samples, and free NNRTI. The polycarboxylated GQD seem to exert the inhibition of RT activity by inhibition of virus-cell binding, in a similar way to what observed for DS8000. The different behavior observed for the GQD sample can be explained by the high


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concentration of nanomaterial required to perform the test (100-fold its IC50) which could allow to the formation of self aggregates in cell culture. In fact, it is widely known that graphene based materials, acting as colloids, are able to remain suspended in solutions by interaction between their functional groups and the chemical counterparts of the suspending media. However, in cell culture media, these nanoparticles can interact with salts, ions and biomolecules thus affording the graphene flocculation or aggregation.

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This effect is absent in the conjugated samples probably because of the presence of the

organic moiety that do not facilitate the nanoparticles aggregation. The hypothesized virus binding activity of GQD further strengthen the hypothesis of a dual mechanism of action for the GQD-CHI499 conjugate system where the easily cleavable imide bond, allows the release of the virus binding inhibitor GQD and of the non-nucleoside reverse transcriptase inhibitor CHI499.

Figure 6. Variation of p24 antigen concentration with time of addition of compounds GQD, GQDCHI499, GQD-CDF119, CHI499 and CDF119, the control/reference compounds DMP266, DS8000 and no-drug control.

CONCLUSIONS


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We have reported here, for the first time, the anti-HIV activity of GQD synthesized by prolonged acidic oxidation of MWCNT and of the same nanomaterial conjugated with the reverse transcriptase inhibitors CHI499 and CDF119. The physicochemical properties of the synthesized GQD-based nanomaterials emerged as relevant features able to control their antiviral activity. The RTI-conjugated compound GQD-CHI499, revealed to be a potential good candidate for HIV treatment showing an IC50 of 0.09 µg/mL, an EC50 value in cell of 0.066 µg/mL and a selectivity index of 362. Time of addiction experiments for both conjugated samples reported a similar mechanism of action to that exerted by the compared reverse transcriptase inhibitors. The conjugate GQD-CHI499 showed a dual mechanism of action exerting the inhibition of RT activity and virus binding to the cells. The reported results could represent a significant starting point for future research on the use of graphene based materials for the HIV therapy. The use of GQD as carrier for HIV inhibitors could open new opportunities in AIDS treatment allowing the administration of poorly water soluble and highly permeable RT inhibitors. Moreover, the surface functionalization of GQD with biocompatible polymers and/or targeting ligands has the potential to further improve their biocompatibility and therapeutic efficacy leading to the development of new anti-HIV agents with an ideal pharmacological profile.

EXPERIMENTAL PROCEDURES Synthesis of CDF119 A solution of 2-{[1-(3,5-dimethylbenzyl)-1H-benzimidazol-2-yl]sulfanyl}-N-2-nitrophenyl acetamide (1 mmol), prepared following a previously reported procedure

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in ethanol (5 mL) was treated with

SnCl2 (7.56 mmol) and the resulting mixture was refluxed for 2h. After completion of the reaction, a 10N aqueous solution of NaOH was added until pH 8 was reached and the mixture was stirred at rt for 1h. The resulting solution was acidified with 2N HCl until pH 5 and the aqueous layer extracted with


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ethyl acetate (3x10ml). The organic layer was dried on anhydrous sodium sulfate. The solvent was then removed under vacuum and the residue was purified by flash chromatography using cyclohexane/ ethyl acetate (60:40) as eluent and crystallized from ethanol. Mp: 141-143 °C, yield 47 %. IR (neat) 3420, 2100, 1640, 1580, 1550, 1430, 1380, 750 cm-1; 1H NMR (DMSO-d6): 2.15 (s, 6H, CH3), 4.85 (s, 2H, CH2), 4.91 (bs, 2H, NH2), 5.31 (s, 2H, CH2), 6.79 (s, 2H, ArH), 6.87 (s, 1H, ArH), 7.15-7.18 (m, 4H, ArH), 7.44-7.63 (m, 3H, ArH), 12.48 (bs, 1H, NH). Anal. Calcd for C24H24N4OS: C: 69.20; H: 5.81; N: 13.45. Found: C: 69.35; H: 5.69; N: 13.62 Synthesis of GQD Pristine MWCNT were reacted with a mixture of HNO3/H2SO4 (1:3 ratio); the mixture was placed in a round bottom flask equipped with a condenser and sonicated for 4 days in an ultrasonic water bath at 60° C. Then, the reaction mixture was diluted with deionized water and filtered under vacuum using a 0,1 µm Millipore membrane; the filtrate was neutralized with NaOH and centrifuged at 3000 rpm. The resulting material was washed with deionized water several times, in order to remove most of the sodium salts and the remaining brown mixture was diluted with deionized water and dialysed for 8 h by using dialysis bags with molecular weight cut-off (MWCO) of 12,000 Da. The resulting material was dried under vacuum at 60° C and used for the next investigations. The amount of acidic groups on the GQD surface was evaluated by titration analysis using the instrument Zetasizer 3000 and was found to be of 2.37 mmol/g.”

Synthesis of GQD-CHI499 To a dispersion of GQD (30 mg) in dichloromethane (10 mL), 0.134 mmol of 1-ethyl-3-(3dimethylaminopropyl)

carbodiimide

hydrochloride

(EDC⋅HCl)

and

0.134

mmol

of

4-

dimethylaminopyridine (DMAP,) were added and the reaction mixture was stirred for 15 min at room temperature

under

argon

atmosphere.

Then,

2-{[1-(3,5-dimethylbenzyl)-1H-benzimidazol-2-


19


yl]sulfanyl}-N-(2-chloro-4-sulfamoylphenyl)-acetamide

(CHI499,

Page 20 of 38

0.134

mmol)

dissolved

in

dichloromethane 5 mL) was added and the mixture was stirred at r. t.. for 4 days. The mixture was centrifuged at 3000 rpm. The residue was diluted with deionized water and dialysed for 8 h by using dialysis bags MW of 12,000 Da. The resulting material was dried at 60° C under vacuum and the amount of drug loaded on the GQD surface was evaluated by TGA analysis under argon atmosphere. The amount of CHI499 loaded on the GQD was found to be of 20%, as evaluated by TGA analysis and evaluated by difference with the amount of drug added to the reaction mixture and that collected from the washing solutions. Synthesis of GQD-CDF119 To a dispersion of GQD (30 mg) in dichloromethane (10 mL), 0.134 mmol of EDC⋅HCl, 0.013 mmol of 1-hydroxybenzotriazole (HOBt) and 0.0134 mmol of DMAP were added and the mixture was stirred at r. t. under argon atmosphere for 1 h. Then, a solution of CDF119 in dichloromethane (5 mL), was added and the suspension was stirred for 4 days at r. t. The mixture was centrifuged at 3000 rpm and the residue was diluted with deionized water and dialysed for 8 h, by using dialysis bags MW of 12,000 Da. The resulting material was dried at 60° C under vacuum and the amount of drug loaded on the GQD surface was evaluated by TGA analysis under argon atmosphere. The amount of CDF119 loaded on the GQD was found to be of 14.5%, as evaluated by TGA analysis and measured by difference with the amount of drug added to the reaction mixture and the amount of drug present in the washing solutions. Biological assays In vitro anti-HIV assay. The antiviral activity of the investigated compounds against HIV was performed in MT-4 cells using the MTT assay as previouslyreported.41 The stock solutions of the tested compounds, prepared to get 10 x final concentration, were added in 25 µl volumes to two series of triplicate wells in order to allow the simultaneous evaluation of their biological effects at the beginning of each experiment, in both mock- and HIV-infected cells. The dispersion of the tested samples (5-fold


20

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dilutions) were directly prepared in flat-bottomed 96-well microtiter trays, using the Biomek 3000 robot (Beckman instruments, Fullerton, CA). The samples of untreated HIV- and mock-infected cell were also included as cell controls. The HIV stock (50 µl) at 100-300 CCID50, which represents the 50 % cell culture infectious doses or culture medium, was added to both the infected or mock-infected wells of the microtiter tray. The mock-infected cells were used in this test in order to evaluate the effects of tested samples on uninfected cells and then to assess their cytotoxicity. The MT-4 cells exponentially growing, were centrifuged at 220 g for 5 minutes and the supernatant was discarded. Then, the MT-4 cells were resuspended at 6 x 105 cells/ml and 50 µl volumes of this solution were transferred to the microtiter tray wells. The viability of both mock-and HIV-infected cells was evaluated spectrophotometrically five days after infection by performing the MTT assay. MTT assay, a colorimetric assay used to assess the cell metabolic activity, is based on the reduction of the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT, Acros Organics) which is yellow colored, by mitochondrial dehydrogenase to the blue-purple colored formazan, whose amount can be spectrophotometrically measured. The related absorbances were measured in an eight-channel computer-controlled photometer (Infinite M1000, Tecan), at the wavelengths of 540 and 690 nm. The obtained data were calculated using the average absorbance value of three wells. CC50 was defined as the 50% cytotoxic concentration of the test samples able to reduce by 50%, the absorbance (OD540) of the mock-infected control. EC50 was defined as the 50% effective concentration able to achieve the 50% of protection against the virus cytopathic effect in the infected cells Reverse transcriptase assay. The recombinant wild type HIV-1 reverse transcriptase (p66/p51), was expressed and purified as previously reported.42 The RT assay was performed using the EnzCheck Reverse Transcriptase Assay kit (Molecular Probes, Invitrogen), as described by manufacturer. The test is based on the quantitative measurement of dsDNA using the PicoGreen dye which after binding to dsDNA or RNA-DNA heteroduplexes, leads to a pronounced increase in fluorescence signal. As reported, the single-stranded nucleic acids produce a weak fluorescence signal increase when it is


21


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applied a sufficiently high dye:base pair ratio.43 This condition was met in the assay. The template poly(rA) of approximately 350 bases, and the oligo(dT)16 primer, have been annealed in a molar ratio of 1:1.2 for 60 min, at room temperature. Then, 52 ng of RNA/DNA was brought into each well of 96well cell culture plate in 20 µl polymerization buffer (60 mMTris-HCl, 13 mM DTT, 60 mM KCl, 8 mM MgCl2, 100 µM dTTP, pH 8.1). The RT enzyme solution (5 µl) were diluted in order to obtain a suitable concentration in the enzyme dilution buffer (50 mM Tris-HCl, 20% glycerol, 2 mM DTT, pH 7.6) and added to the reaction mixtures, which were incubated for 40 minutes at 25°C and then stopped by the addition of EDTA (15 mM fc). The heteroduplexes were then detected by addition of PicoGreen reagent. The fluorescence signals were read using the excitation wavelength of 490 nm and the emission detection at 523 nm, by using a spectrofluorometer (Safire 2, Tecan). In order to evaluate the activity of the samples against RT, the compounds (1 µl in DMSO) were added to each well before the addition of RT solution. The control wells used as control, contained the same amount of DMSO. The obtained results were expressed as relative fluorescence, i.e. the reaction signal of the mixture in the presence of the tested samples divided by the fluorescence signal of the same reaction mixure without the investigated compounds. Time-of-Addition Experiments: TOA experiments were performed following a previously reported procedure.39 MT-4 cells, infected with HIV-1(IIIB) at 0.5 multiplicity of infection, were treated with the investigated samples by addition at different times (0, 1, 2, 3, 4, 5, 6, 7, 8, 25, and 26 h) after the HIV infection The production of p24 antigen was measured 31 h post infection by the p24 antigen enzymelinked immunosorbent assay. The investigated samples were added at standard concentrations, i.e. 100 times their EC50 value, which represents the concentration required to reduce by 50% the cytopathicity of HIV-1(IIIB) in MT-4 cells.

SUPPORTING INFORMATION


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The contents of Supporting Information include: Materials, chemical, physical and morphological characterization, XRD spectra of MWCNT and GQD, PL spectra of GQD, Raman spectra of MWCNT and GQD, scheme of CDF119 synthesis.

ACKNOWLEDGMENTS The authors are grateful to Prof Salvatore Patanè and Dr Claudia Triolo, Department of Mathematics, Computer Sciences, Physics and Earth Sciences of the University of Messina, for their support in performing atomic force microscopy (AFM) images.

REFERENCES 1. Alvarez, M. M., Aizenberg, J., Analoui, M., Andrews, A. M., Bisker, G., Boyden, E. S., Kamm, R. D., Karp, J. M., Mooney, D. J., Oklu, R. et al. (2017) Emerging Trends in Micro- and Nanoscale Technologies in Medicine: From Basic Discoveries to Translation. ACS Nano 11, 5195−5214. 2. Ma, W., Cheetham, A. G. and Cui, H. (2016) Building nanostructures with drugs. Nano Today, 11, 13−30. 3. Karimi, M., Ghasemi, A., Zangabad, P.S., Rahighi, R., Basri, S.M.M., Mirshekari, H., Amiri, M., Pishabad, Z.S., Aslani, A., Bozorgomid, M. et al. (2016) Smart micro/nanoparticles in stimulus-responsive drug/gene delivery systems. Chem. Soc. Rev. 45, 1457–1501. 4. McNeil, S. E. (2011) Unique benefits of nanotechnology to drug delivery and diagnostics. Methods Mol. Biol., 697, 3–8.


23


Page 24 of 38

5. Sued, O., Figuero, M. I. and Cahn, P. (2016) Clinical challenges inHIV/AIDS: Hints for advancing prevention and patient management strategies. Adv. Drug Deliv. Rev., 103, 5-19. 6. Richman, D. D., Margolis, D. M., Delaney, M., Greene, W. C., Hazuda, D. and Pomerantz, R. J. (2009) The challenge of finding a cure for HIV infection. Science, 323, 1304–1307. 7. Puhan, M. A., Van Natta, M. L., Palella, F. J., Addessi, A. and Meinert, C. (2010) Excess mortality in patients with AIDS in the era of highly active antiretroviral therapy: temporal changes and risk factors. Clin. Infect. Dis. 51, 947–956. 8. Rao, K. S., Ghorpade, A- and Labhasetwar, V. (2009) Targeting anti-HIV drugs to the CNS. Expert Opin. Drug Deliv. 6, 771–784. 9. Mamo, T., Moseman, E. A., Kolishetti, N., Salvador-Morales, C., Shi, J., Kuritzkes, D. R., Langer, R., Von Andrian, U. and Farokhzad. O. C. (2010) Emerging nanotechnology approaches for HIV/AIDS treatment and prevention. Nanomedicine 5, 269–285. 10. J. Lisziewicz, E.R Toke, Nanomedicine applications towards the cure of HIV (2013). Nanomedicine 9, 28–38. 11. Iannazzo, D., Piperno, A., Pistone, A., Grassi, G. and Galvagno, S. (2013) Recent Advances in Carbon Nanotubes as Delivery Systems for Anticancer Drugs. Curr. Med. Chem. 20, 1333– 1354. 12. Martinez, Z. S., Castro, E., Seong, C.-S., Cerón, M. R., Echegoyen, L. and Llano, M. (2016) Fullerene Derivatives Strongly Inhibit HIV-1 Replication by Affecting Virus Maturation without Impairing Protease Activity. Antimicrob Agents Chemother. 60, 5731-5741. 13. Kornev, A. B., Peregudov, A. S., Martynenko, V. M., Guseva, G. V., Sashenkova, T. E., Rybkin, A. Y., Faingold, I. I., Mishchenko, D. V., Kotelnikova, R. A., Konovalova, N. P. et al. (2013)


24

Page 25 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Synthesis and biological activity of a novel water-soluble methano[60] fullerene tetracarboxylic derivative. Mendeleev Commun. 2013, 23, 323–325. 14. Zeng, S., Zhou, G., Guo, J., Zhou, F. and Chen, J. (2016) Molecular simulations of conformation change and aggregation of HIV-1 Vpr13-33 on graphene oxide. Sci. Rep. 6, 24906 (7pp). 15. Zhang, M., Mao, X., Wang, C., Zeng, W., Zhang, C., Li, Z., Fang Y., Yang, Y., Liang W. and Wang, C. (2013) The effect of graphene oxide on conformation change, aggregation and cytotoxicity of HIV-1 regulatory protein (Vpr). Biomaterials, 34, 1383–1390. 16. Liu, Z., Winters, M., Holodniy, M. and Dai, H. (2007) SiRNA delivery into human T cells and primary cells with carbon-nanotube transporters. Angew. Chem. Int. Ed. 46, 2023–2027. 17. Iannazzo, D., Pistone, A., Galvagno, S., Ferro, S., De Luca, L., Monforte, A.M., Da Ros, T., Hadad, C., Prato, M., Pannecouque, C. (2015) Synthesis and anti-HIV activity of carboxylated and drug-conjugated multi-walled carbon nanotubes. Carbon 82, 548–561. 18. Krishnaraj, R. N., Chandran, S., Pal, P. and Berchmans, S. (2014) Investigations on the antiretroviral activity of carbon nanotubes using computational molecular approach. Comb. Chem. High Throughput Screen.17, 531-535. 19. Iannazzo, D., Ziccarelli, I. and Pistone, A. (2017) Graphene quantum dots: multifunctional nanoplatforms for anticancer therapy. J. Mat. Chem. B 5, 6471–6489. 20. Yan, X., Zhao, X. E., Sun, J., Zhu, S., Lei, C., Li, R., Gong, P., Lin, B., Wang, R., Wang, H. (2018) Probing glutathione reductase activity with graphene quantum dots and gold nanoparticles system. Sensor Actuat. B-Chem, 263, 27–35. 21. Gao, Y., Yan, X., Li, M., Gao, H., Sun, J., Zhu, S., Han, S., Jia, L.-N., Zhao, X.-E., Wang, H. (2018) A “turn-on” fluorescence sensor for ascorbic acid based on graphene quantum dots via


25


Page 26 of 38

fluorescence resonance energy transfer. Anal. Methods, 10, 611–616. 22. Zhao, X.E., Lei, C., Gao, Y., Gao, H., Zhu, S., Yang, X., You J., Wang, H. (2017) A ratiometric fluorescent nanosensor for the detection of silver ions using graphene quantum dots. Sensor Actuat. B-Chem.,253, 239–246. 23. Zhu, S., Meng, Q., Wang, L., Zhang, J., Song, H., Jin, H., Zhang, K., Sun, H., Wang, H. and Yang, B. (2013) Highly photoluminescent carbon dots for multicolor patterning sensors, and bioimaging. Angew. Chem. Int. Ed. 52, 3953–3957. 24. Chen, M. L., He, Y. J., Chen, X.W. and Wang, J. H. (2013) Quantum-dot-conjugated graphene as a probe for simultaneous cancer-targeted fluorescent imaging, tracking, and monitoring drug delivery. Bioconjugate Chem. 24, 387–397. 25. Iannazzo, D. Pistone, A. and Galvagno, S. (2015) Functionalization methods of graphene. Chemical Functionalization of Carbon Nanomaterials: Chemistry and Applications (Thakur, V.K., Thakur, M.K. Eds.), pp. 510–537, Chapter 21.CRC Press, Boca Raton (FL). 26. Fahmi, M. Z., Sukmayani, W., Khairunisa, S. Q., Witaningrum, A. M., Indriati, D. W., Matondang, M. Q. Y., Chang, J.-Y., Kotakie T. and M. Kameoka (2016) Design of boronic acidattributed carbon dots on inhibits HIV-1 entry. RSC Advances 6, 92996–93002. 27. Monforte, A. M., Logoteta, P., Ferro, S., De Luca, L., Iraci, N., Maga, G., De Clercq, E., Pannecouque, C. and Chimirri, A. (2009) Design, synthesis, and structure-activity relationships of 1,3-dihydrobenzimidazol-2-one analogues as anti-HIV agents. Bioorg. Med. Chem. 17, 5962– 5967. 28. Monforte, A. M., Logoteta, P., De Luca, L., Iraci, N., Ferro, S., Maga, G., De Clercq, E., Pannecouque, C. and Chimirri, A. (2010) Novel 1,3-dihydro-benzimidazol-2-ones and their analogues as potent non-nucleoside HIV-1 reverse transcriptase inhibitors. Bioorg. Med. Chem. ACS Paragon Plus Environment

26

Page 27 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


18, 1702–1710. 29. Iannazzo, D., Pistone, A., Salamò, M., Galvagno, S., Romeo, R., Giofré, S. V., Branca, C., Visalli, G., Di Pietro, A. (2017). Graphene quantum dots for cancer targeted drug delivery. Int. J. Pharm. 518, 185–192 30. Lavin, J. G., Subramoney, S., Ruoff, R., Berber. S. and Tomanek, D. (2002) Scrolls and nested tubes in multiwall carbon nanotubes. Carbon 40, 1123–1130. 31. Lin, L. and Zhang, S. (2012) Creating high yield water soluble luminescent graphene quantum dots via exfoliating and disintegrating carbon nanotubes and graphite flakes. Chem. Commun. 48, 10177–10179. 32. Chen, W., Yan, L. and Bangal, P.R. (2010) Chemical reduction of graphene oxide to graphene by sulfur-containing compounds. J. Phys. Chem. C 114, 19885–19890. 33. Chua, C. K., Sofer, Z., Šimek, P., Jankovsky´, O., Klímová, K., Bakardjieva, S., Kučkovà, S. H. and Pumera, M. (2015) Synthesis of Strongly Fluorescent Graphene Quantum Dots by CageOpening Buckminsterfullerene. ACS Nano 9, 2548–2555. 34. Bianco, A., Kostarelos, K. and Prato, M. (2011) Making carbon nanotubes biocompatible and biodegradable. Chem. Commun., 47, 10182–10188. 35. Prakash, S., Mishra, R., Malviya, R. and Sharma, P. K. (2014) Measurement Techniques and Pharmaceutical Applications of Zeta Potential: A Review. J. Chronother. Drug Deliv. 5, 33–40. 36. Monforte, A. M., Ferro, S., De Luca, L., Lo Surdo, G., Morreale, F., Pannecouque, C., Balzarini, J. and Chimirri, A. (2014) Design and synthesis of N1-aryl-benzimidazoles 2-substituted as novel HIV-1 non-nucleoside reverse transcriptase inhibitors. Bioorg. Med. Chem. 22, 1459–1467. 37. Huang, C. L., Huang, C. C., Mai, F. D., Yen, C. L., Tzing, S. H., Hsieh, H. T., Ling Y. C. and


27


Page 28 of 38

Chang, J.Y. (2015) Application of paramagnetic graphene quantum dots as a platform for simultaneous dual-modality bioimaging and tumor-targeted drug delivery, J. Mater. Chem. B 3, 651–664. 38. Witvrouw, M., Fikkert, V., Pluymers, W., Matthews, B., Mardel, K., Schols, D., Raff, J., Debyser, Z., De Clercq, E., Holan, G. et al. (2000) Polyanionic (i.e., Polysulfonate) Dendrimers Can Inhibit the Replication of Human Immunodeficiency Virus by Interfering with Both Virus Adsorption and Later Steps (Reverse Transcriptase/Integrase) in the Virus Replicative Cycle. Mol. Pharmacol 58, 1100–1108. 39. Daelemans, D., Pauwels R., De Clercq, E. and Pannecouque, C. (2011) A time-of–drug addition approach to target identification of antiviral compounds. Nature Protocols 6, 925–933. 40. McCallion, C., Burthem, J., Rees-Unwin, K., Golovanov, A., Pluen, A. (2016) Graphene in therapeutics delivery: Problems, solutions and future opportunities. Eur. J. Pharm. Biopharm. 104, 235–250. 41. Balzarini, J., Karlsson, A., Perez-Perez, M. J., Vrang, L., Walbers, J., Zhang, H., Oberg, B., Vandamme, A. M., Camarasa, M. J. and De Clercq, E. (1993) HIV-1-specific reverse transcriptase inhibitors show differential activity against HIV-1 mutant strains containing different amino acid substitutions in the reverse transcriptase. Virology 192, 246–253. 42. Auwerx, J., North, T. W., Preston, B. D., Klarmann, G. J., De Clercq, E. and Balzarini, J. (2002) Chimeric human immunodeficiency virus type 1 and feline immunodeficiency virus reverse transcriptases: role of the subunits in resistance/sensitivity to nonnucleoside reverse transcriptase inhibitors. Mol. Pharmacol. 61, 400–406. 43. Singer, V. L., Jones, L. J., Yue, S. T. and Haugland, R. P. (1997) Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA


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quantitation. Anal. Biochem. 249, 228–238.


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Table of Contents

Graphene Quantum Dots based systems as HIV Inhibitors Daniela Iannazzo, Alessandro Pistone, Stefania Ferro, Laura De Luca, Anna Maria Monforte, Roberto Romeo, Maria Rosa Buemi and Christophe Pannecouque


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Figure 1. GQD and GQD-RTI conjugates. 132x100mm (300 x 300 DPI)



Figure 2. (A) Representative HRTEM images of GQD sample; (B) Intensity of size distribution of GQD sample dispersed in deionized water at concentration of 0.1 mg/mL; (C) Isoelectric titration graph of GQD sample. The pH of medium dependence of zeta potential was evaluated at concentration of GQD of 0.1 mg/mL. 321x281mm (96 x 96 DPI)


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Scheme 1. Synthesis of GQD-CHI499 and GQD-CDF119. Reagents and conditions: (a) CHI499, EDC⋅HCl, DMAP, CH2Cl2, r. t., 4 d; (b) CDF119, EDC⋅HCl, HOBt, DMAP, CH2Cl2, r. t., 4 d. 217x150mm (300 x 300 DPI)



Figure 3. (A) FTIR spectra of GQD, GQD-CHI499, and GQD-CDF119; (B) TGA curves for GQD, GQD-CHI499 and GQD-CDF119. All the experiments were performed under argon atmosphere. 356x150mm (96 x 96 DPI)


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Figure 4. PL spectra of GQD, GQD-CHI499, and GQD-CDF119. 375x266mm (96 x 96 DPI)



Figure 5. Dispersibility of GQD, GQD-CHI499 and GQD-CDF119 in pure water (A) and PBS (C) and calculation of the corresponding zeta potential values in pure water (B) and PBS (D). 196x166mm (150 x 150 DPI)


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Figure 6. Variation of p24 antigen concentration with time of addition of compounds GQD, GQD-CHI499, GQD-CDF119, CHI499 and CDF119, the control/reference compounds DMP266, DS8000 and no-drug control. 478x202mm (96 x 96 DPI)



355x169mm (96 x 96 DPI)


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Graphene Quantum Dots Based Systems As HIV Inhibitors

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