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-Cyclodextrin-grafted on multiwalled carbon nanotubes as versatile nanoplatform for entrapment of guanine-based drugs Daniela Iannazzo a,1 , Antonino Mazzaglia b,∗,1 , Angela Scala b,1 , Alessandro Pistone a,1 , Signorino Galvagno a , Maurizio Lanza c , Cristina Riccucci d , Gabriel Maria Ingo d , Ivana Colao e , Maria Teresa Sciortino e , Francesco Valle f , Anna Piperno g,∗∗ , Giovanni Grassi g a Dipartimento di Ingegneria Elettronica, Chimica e Ingegneria Industriale, Università di Messina, C.da di Dio, 98166 Messina, Italy b Consiglio Nazionale delle Ricerche CNR-ISMN c/o Dip. di Scienze Chimiche dell’Università di Messina, Viale Ferdinando Stagno D’Alcontres 31, 98166 Messina, Italy c Consiglio Nazionale delle Ricerche CNR-IPCF, Viale Ferdinando Stagno D’Alcontres, 98166 Messina, Italy d Consiglio Nazionale delle Ricerche CNR-ISMN, UOS Montelibretti, Via Salaria, 00015 Monterotondo Stazione, Rome, Italy e Dipartimento di Scienze Biologiche e Ambientali, Università di Messina, Viale Ferdinando Stagno D’Alcontres, 98166 Messina, Italy f Consiglio Nazionale delle Ricerche CNR-ISMN, Via Piero Gobetti 101, 40129 Bologna, Italy g Dipartimento di Scienze Chimiche, Università di Messina, Viale Ferdinando Stagno D’Alcontres 31, 98166 Messina, Italy
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Article history: Received 30 May 2014 Received in revised form 1 September 2014 Accepted 14 September 2014 Available online xxx Keywords: Click chemistry Multiwalled carbon nanotubes -Cyclodextrins Guanine Acyclovir
a b s t r a c t The design of -cyclodextrin/multiwalled carbon nanotubes hybrid (-CD-MWCNT) as nanoplatform for the entrapment and delivery of guanine based drugs is described here. The functionalized carbon nanomaterials have been characterized by XPS spectroscopy, electron microscopy (FEG-SEM and TEM), AFM, TGA, and FT-IR to achieve insights on structure, morphology and chemical composition. The drug binding abilities of nanocarrier towards the guanine (G) and Acyclovir (Acy) were proved by UV–vis and DSC experiments. Host–guest equilibrium association constants and drug loading have been evaluated for G/-CD-MWCNT and Acy/-CD-MWCNT complexes. The release studies showed a sustained delivery of Acy without initial burst effect confirming a strong interaction of drug with the nanoplatform sites. The preliminary antiviral data indicated that the Acyclovir loaded into the -CD-MWCNT platform interferes with HSV-1 replication and the antireplicative effect was higher than the free drug. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Over the last decade, functionalized carbon nanotubes (f-CNT) are emerging as promising delivery systems for the transport and release into the cells of small drugs, nucleic acids or proteins [1–4]. Chemical modifications on CNT, changing the physicochemical nature of CNT dispersions (CNT individualization against small or large bundles), allow to overcome the complete aqueous insolubility of unmodified CNT (pristine) and decrease their potential risk from medical use. To improve the CNT dispersibility in a biological
∗ Corresponding author. Tel.: +39 0903974108; fax: +39 0903974108. ∗∗ Corresponding author. Tel.: +39 090 6765173; fax: +39 090 393897. E-mail addresses: [email protected] (A. Mazzaglia), [email protected] (A. Piperno). 1 These authors contributed equally to this work.
milieu, several non-covalent and covalent chemical modification methods have been explored and there is increasing evidence that biocompatibility and biodegradation of CNT can be achieved by appropriate functionalization [5]. Moreover, the nature of the groups, linked covalently or by supramolecular approach to the CNT surface, affects the pathways of cellular internalization, i.e. passive diffusion by means of “nanoneedle” or energy-dependent cellular uptake [6]. CNT surface modification by macrocyclic host molecules, such as crown ethers [7,8], calixarenes [9], cyclodextrins [10–13], cucurbiturils [14], has gained significant attention because of their important roles in supramolecular chemistry. Among a wide range of macrocyclic molecules, -cyclodextrins (CD), cyclic oligosaccharides based on seven d-glucose units linked by ␣-1,4 glucose bond, are well established drug carriers with high biocompatibility able to host different guest molecules by inclusion into the cavity or interaction with external rims. The combination of the physical properties of CNT and the molecular recognition ability
http://dx.doi.org/10.1016/j.colsurfb.2014.09.025 0927-7765/© 2014 Elsevier B.V. All rights reserved.
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of -CD grafted on the CNT surface generates interesting constructs with promising applications in different fields such as environment [15], catalysis [16], chemical sensing [17]. Although different synthetic procedures have been proposed to obtain cyclodextrin grafted-CNT [15–20], at the best of our knowledge, no data are available regarding the effectiveness of -CD-CNT covalently conjugates as delivery system able to sustain the release of small drugs. Our ongoing programme is aimed for the discovery of new antiviral drugs [21–26], with enhanced therapeutic efficiency and reduced unwanted side effects. In this direction our interest was focused on the synthesis of a new nanocarrier-based delivery system for the multimodal anchorage of antiviral drugs. Here, we report an approach to fabricate -cyclodextrin/multiwalled carbon nanotubes (-CD-MWCNT) nanohybrid as prospective platform for drug delivery. The present work newly describes the synthesis of the nanohybrid by click-chemistry, characterization by complementary techniques and recognition ability of the system towards the guanine, a poor soluble DNA base. Afterwards, the interaction of the nanohybrid with Acyclovir (Acy), the prototype of acyclic nucleoside analogues, active on three human alpha herpesvirus (HSV-1, HSV-2 and VZV) has been investigated. In our study, Acy was selected as hydrophilic antiviral drug model, in virtue of the easy reliability of its biological assay and for its physical properties that cause poor oral bioavailability. For improving the efficacy of Acy treatment and decreasing its adverse side effects, many technological approaches comprising pro-drug preparations, innovative formulations and subcutaneous delivery system that provide a long-lasting sustained release have been proposed [27,28]. The release studies of Acy loaded on our nanoplatform showed a slow release, about 15% after 8 days, without the initial burst effect. The preliminary antiviral data indicated that Acy/-CD-MWCNT complex interferes with HSV-1 replication and the antireplicative effect is higher than the free drug. As proof of principle, this study has demonstrated the reliability of -cyclodextrin/multiwalled carbon nanotubes nanohybrid as attractive platform for the loading and delayed release of drugs. 2. Materials and methods 2.1. Materials MWCNT were produced by catalytic chemical vapor deposition (CCVD) from isobutane on Fe/Al2 O3 catalyst [29]; then synthesized MWCNT were subjected to purification and oxidation reaction to obtain carboxylated multiwalled carbon nanotubes (MWCNT-Ox) as previously reported [30]. All other reagents were of the highest commercial grade available and were used as received or were purified by distillation or re-crystallization when necessary. All the solutions used for spectroscopic characterization were prepared in ultrapure microfiltered water (Galenica Senese) and analysed at 298 K. 2.2. Preparation of MWCNT-alkyne (3) MWCNT-Ox (1) [30] (400 mg) were dispersed in odichlorobenzene (100 mL), sonicated for 15 min at room temperature; then a solution of p-(2-propylnyloxy)-benzamine (2) [20] (2 g dissolved in 200 mL of dry acetonitrile) was added, and the mixture was sonicated for 30 min at room temperature. The reaction mixture was stirred for 10 min under helium flow, added with 3.0 mL of isoamyl nitrite and stirred at 60 ◦ C for 24 h under helium flow. The mixture was then cooled, diluted with ethanol and filtered under vacuum on Millipore membrane of 0.1 m. The solid residue was washed with water, ethanol, chloroform
and ethyl ether (3 × 50 mL) and each time sonicated for 5 min. The residue was dried under vacuum at 50 ◦ C to give 402 mg of MWCNT-alkyne (3). From TGA analysis the amount of grafted alkyne moieties onto the surface of MWCNT could be estimated to be ∼5 wt% corresponding to ∼0.09 mmol g−1 . 2.3. Preparation of ˇ-CD-MWCNT nanohybrid (5) 100 mg of (3) were dispersed in 15 mL of dry and degassed DMF. The mixture was sonicated for 15 min under helium flow and then treated with 125 mg of mono-6-deoxy-6-azido--cyclodextrin (4) [31] (125 mg, 0.125 mmol), CuSO4 (15 mg, 0.085 mmol) and Na ascorbate (35 mg, 0.17 mmol). The reaction mixture was sonicated for 10 min and heated at 85 ◦ C for 48 h under helium flow; then cooled to room temperature, diluted with ethyl acetate (50 mL) and filtered under vacuum on Millipore membrane of 0.1 m. The solid residue was washed with water, ethanol, and ethyl ether (3 × 50 mL) and each time sonicated for 5 min. The residue was dried under vacuum at 50 ◦ C to give 70 mg of -CD-MWCNT (5). From TGA analysis the amount of -CD grafted onto the surface of MWCNT could be estimated to be ∼6 wt% corresponding to ∼0.055 mmol g−1 . 2.4. Investigation of drugs/ˇ-CD-MWCNT interaction and complexes preparation 2.4.1. Interaction of ˇ-CD-MWCNT with G A stock dispersion of nanohybrid (5) has been prepared by dispersing -CD-MWCNT (1 mg/mL) in ultrapure water and sonicating the mixture for 2 min. A stock solution of 600 M of guanine (G) in MeOH has been prepared and various aliquots of 200 L of this solution have been evaporated. Organic films of G have been re-dissolved, by increasing amounts of nanohybrid aqueous dispersions (from 15 to 60 M) at different [G]//[-CD-] molar ratios. In all the systems [G] was 60 M. 2.4.2. Interaction of ˇ-CD-MWCNT with Acy The Acy/-CD-MWCNT interaction was investigated by titrating an aqueous solution of Acy (60 M) with increasing amounts of an aqueous dispersion of -CD-MWCNT (1 mg/mL). -CD-MWCNT has been sonicated for 2 min, and added from 15 to 60 M at different [Acy]//[-CD-] molar ratios. All the systems have been stirred at 298 K for 2 days, equilibrated for 1 day at r.t. and then analysed. In all the samples the concentration of host nanohybrid is given as in -CD units [-CD-] grafted onto the surface of MWCNT, as evaluated by TGA (∼ =0.055 mmol g−1 ). The G/MWCNT complex was prepared with the same procedure used for G/-CD-MWCNT complexes. Pristine MWCNT (0.7 mg/mL) was dispersed under 2 min sonication in ultrapure water. Competition experiments were carried out by adding an excess of (∼ =20 eq) of 1-adamantanol (ADA) to the complex [G]/[-CD-] 1:0.5 molar ratio. The complex was preventively centrifuged, the supernatant was removed and the residue was re-dispersed in aqueous solution. After adding the ADA, the dispersion was mixed by vortex and analysed freshly or upon storage at room temperature. The association constants have been estimated according to the equation of Benesi and Hildebrand (double-reciprocal method) [32]. 2.5. Drug loading The G/-CD-MWCNT complex and the Acy/-CD-MWCNT complex at 10:1 molar ratio were centrifuged with 2000 r min−1 for 6 min and repeatedly filtered with a Millipore membrane of 0.22 m, until free, unbound drugs were no more detected.
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The precipitates were re-dispersed in 2 mL of ultrapure microfiltered water and UV–vis spectra were recorded. On the basis of optical absorbance data and molar extinction coefficients (ε247 ∼ = 1670 M−1 cm−1 ; ε252 ∼ = 11,400 M−1 cm−1 , for G and Acy respectively), a 6.5% and 12.2% drug loading was calculated for guanine and Acy, respectively, using the following equation: Drug loading = (drug weight in the nanoparticles/weight of the nanoparticles) × 100. 2.6. In vitro release study The in vitro release experiments were performed in 10 mM phosphate buffer containing NaCl (137 mM) and KCl (2.7 mM) at pH 7.4 (PBS) by a dialysis method. A known amount of the 1:1 complex (2.5 mg) was dispersed in 2.5 mL of PBS and placed in a dialysis bag (MWCO = 3500 Da, Spectra/Por® ). The sample was plunged in PBS of higher volume (15 mL) under stirring (sink condition) and kept at 37 ◦ C. At fixed times, 1 mL of release medium was withdrawn and replaced with an equal volume of fresh and analysed by UV–vis by using ε252 . The release of free Acy was carried out using 2.5 mL of a stock solution of Acy (1.4 mg in 100 mL PBS) in the inner bag and 15 mL of PBS in the external bag. All the experiments were carried out in triplicate. 2.7. Cytotoxicity and plaque reduction assay Vero cells were collected and cytotoxicity was evaluated by Trypan blue exclusion standard assay (Sigma–Aldrich). Percentage of death cells was evaluated in a Burker chamber using 20× magnification of an inverted microscope. The virus was diluted to yield 30 plaques/100 L. All the volumes were inoculated on monolayers of Vero cells in 24-well dishes and incubated for 1 h at 37 ◦ C. After the incubation time, the inoculum was removed and the monolayers were covered with Dulbecco’s-modified Eagle’s medium containing 0.3% methylcellulose included compounds at different concentrations (60, 6 and 0.6 M). After 3 days of incubation at 37 ◦ C, the medium was aspirated and the cells were then fixed, stained with crystal violet, and visualized at 10× magnification with an inverted microscope for plaque detection. The assays were performed in duplicate for each dilution. 3. Results and discussion 3.1. Synthesis and characterization of ˇ-CD-MWCNT As substrate for the synthesis of -CD-MWCNT nanohybrid (Fig. 1), we have selected the carboxylated multiwalled carbon nanotubes (MWCNT-Ox), which did not show cytotoxicity in our previous studies [30,33]. MWCNT-Ox obtained by oxidation of pristine MWCNT contains 1.8 mmol g−1 of free OH groups of which about 1.2 mmol g−1 are carboxylic group available for the chemical derivatization [30,33]. Our approach relies on the derivatization of MWCNT-Ox (1) with p-(2-propylnyloxy)benzammine (2), using diazotization-coupling procedure, to obtain (3). Alkyne-functionalized MWCNT (3) has been successfully coupled by click reaction with the monoazide--CD (4) (-CD-N3 ), giving the -CD-MWCNT nanohybrid (5), through Cu-mediated acetylene-azide coupling (CuAAC) reaction (Huisgen cycloaddition) using CuSO4 /Na-ascorbate as metal source. The functionalized carbon nanomaterials have been characterized by XPS, microscopy (FEG-SEM, TEM, AFM), TGA and FT-IR to gain insights on structure, morphology and chemical composition. XPS spectra of pristine MWCNT showed only an intense C 1s peak [34] at a binding energy ca. 284 eV, while for the all functionalized CNT, the detection of a N 1s peak at ca. 400 eV and O 1s peak at ca. 532 eV indicated the success of the functionalization processes (see Fig. 1 SM). According to
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our previous results [35], MWCNT-Ox XPS spectra show the presence of a N 1s peak at ca. 400 eV indicating that the oxidative process is not selective towards the formation of carboxylic groups, but also some other nitrogenated chemical groups are introduced. XPS data provide an estimate of the functionalization degree (m), via the comparison of the amount of nitrogen and carbon (N:C ratio), and oxygen and carbon (O:C ratio), in agreement with the m calculated by TGA. The m of MWCNT-Ox (1), MWCNT-alkyne (3) and -CD-MWCNT (5) was deduced comparing the TGA data obtained by thermal decomposition of the surface groups heated up to 700 ◦ C under inert atmosphere and was found of 18%, 23% and 29% respectively (Fig. 1). According to literature data [36,37], the TGA curve for -CD-MWCNT (5) exhibits two steps of weight loss, contributed from the loss of residual water (in the range 30–190 ◦ C) and the loss of -CD in the range of 200–450 ◦ C, as shown by DTG curve (inset of Fig. 1). Thus, the amount of -CD grafted onto the surface of MWCNT could be estimated to be ∼6 wt% corresponding to ∼0.055 mmol g−1 . The FT-IR spectra (see Fig. 2 SM) indicated the successful covalent grafting of the -CD moiety on the MWCNT. For oxidized MWCNT (1) the curve shows the peaks at 3445 (O H vibration), 2930 (C H stretching), 1710 and 1150 (C O and C O stretching), 1630 (C C stretching) cm−1 . After diazotization-coupling, the IR spectrum of alkyne modified MWNT (3) exhibits the characteristic absorption of alkyne group at 2130 cm−1 . The characteristic peaks due to the strong absorptions of C O stretching vibration of -CD at 1220, 1170, 1100 and 1030 cm−1 can be clearly seen in the IR spectrum of the nanohybrid (5). FEG-SEM analyses showed a high degree of bundling for MWCNT-Ox (1), while in -CD-MWCNT (5) carbon nanotubes appear entangled together inside a smooth matrix. This topographic aspect can be explained by partial cross-linking of the carbon nanotubes and the -cyclodextrin with this latter homogeneously deposited along the nanotube shaping densely packed carbon nanotubes (see Fig. 3 SM). TEM images of pristine MWCNT showed a clear multi-walled tube structure with a diameter close to 15–20 nm [34]. After the acid treatment, MWCNT-Ox (1) are dispersed and shortened on account of the severe oxidation of nitric–sulphuric acid mixture (see Fig. 4a SM). The external walls of MWCNT-Ox appear severely eroded with several continuous graphite layers often truncated and significant faults between graphite carbon planes; no amorphous layers on the external walls of MWCNT-Ox were observed (see Fig. 4b SM). TEM analyses confirmed the success of the grafting of cyclodextrin to carbon nanotubes; the -cyclodextrin molecules densely decorate the carbon nanotubes (see Fig. 4c SM) and a diffuse hazy contrast on the external walls (indicated by arrow), due to linkage with amorphous -cyclodextrin moieties, was observed (see Fig. 4d SM). AFM images (see Fig. 5a–c SM) have allowed to characterize the morphology of the nanotubes both bare and functionalized. It is noteworthy that when dealing with nanometer sized objects the parameter that is less affected by the tip convolution is the height, because the lateral size is usually enlarged and influenced because of the tip shape. The functionalization of MWCNT with -CD leads to an increase in the lateral nanotube size that is particularly well appreciated in the section analysis where a net height increase is evident. -CD-MWCNT nanohybrid exhibits a multiwalled tube structure of 15–20 layers with an average length of 200–1000 nm. The height was found to be about 5 nm, as determined by AFM analysis (see Fig. 5c SM). 3.2. Complexation capability towards guanine-based drugs In order to probe the recognition ability of -CD-MWCNT nanohybrid, guanine (G), a poor soluble DNA base, and Acyclovir, an
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Fig. 1. Right: preparation of -CD-MWCNT nanohybrid (5) with the values of m% 700 ◦ C, N/C ratio and O/C ratio obtained from TGA and XPS data. Left: TGA profile of MWCNT and 1, 3 and 5; in the inset DTG curve of 5.
acyclic analogue of the natural nucleoside 2 -deoxyguanine, were selected as molecular models in the complexation experiments. Studies concerning the DNA bases interactions with cyclodextrin modified carbon nanotubes are rare, except for few reports [17,38], which suggest that purine bases have a strong affinity towards the CD cavity. In our study, the complexation capability of -CD-MWCNT towards G was investigated by UV–vis. Fig. 2A shows the UV–vis spectra of G in ultrapure water containing various amounts of -CD-MWCNT (from 1:0.25 to 1:1 [G]/[-CD-] molar ratio). It is clear that the UV–vis spectrum of G changes with the addition of -CD-MWCNT. In particular, by increasing the amount of -CD-MWCNT, at the highest molar ratio (1:0.25), the UV–vis spectrum (Fig. 2A, curve b) shows hypocromicity of the G bands centred at 247 and 278 nm, presumably due to G present in two different environments, ascribable to free G in equilibrium with G allocated on -CD-MWCNT. The propensity of purine base to interact with nanohybrid is more evident at lower molar ratio (1:0.5; Fig. 2A, curve c), as shown by changes of the peculiar double band whose maxima were red-shifted from 247 to 253 nm and blueshifted from 278 to 273 nm, respectively. At 1:1 molar ratio (Fig. 2A, curve d), only a wide band (with a maximum at 270 nm and a shoulder at 256 nm) appears and this spectral change was tentatively ascribed to a strong interaction of G with the nanohybrid. The modifies in the UV–vis spectra can plausibly depend on the several interactions of G with the different platform sites, comprising the selective inclusion into the CD cavity and the stacking [12] on the CNT surface and/or on 1,2,3-triazole ring and phenyl
group of the linker. With the aim to characterize G/nanohybrid species, we underlined the spectral changes of G by increasing the amount of nanohybrid, in a selected range of [G]//[-CD-] molar ratios (from 1:0.3 to 1:0.6, see Fig. 2B). As anticipated, no shift of absorption maxima has been observed till about 1:0.4 molar ratio. On the other hand at lower molar ratios (from 1:0.5 molar ratio), evident wavelength shifts have been observed and a slight recovery of absorption intensity. In detail, the presence of a nearly-isosbestic point between 24 and 36 M in -CD units, implies an equilibrium between different species. To elucidate the prominent interaction sites of G and investigate the role of the CD cavity in the complexation process, 1-adamantanol (ADA) has been chosen as a competitive guest for G for its ability to strongly interact into the inner cavity of CD, not possessing bands in the absorption region of DNA base. Fig. 3 shows the UV–vis spectra of G/-CD-MWCNT complex (prepared at 1:0.5 [G]/[-CD-] molar ratio, curve c) after addition of ADA. Spectra of ADA/-CD-MWCNT/G were registered on fresh sample after few minutes, and after 1 month (curves c and c , respectively). We hypothesize an ADA involvement in the complexation equilibrium, probably moving G from the hydrophobic cavity of the CD to different recognition sites i.e. CNT surface and/or to the triazole and phenyl groups. The UV–vis spectra registered after 1 month clearly indicate a noticeable change referable to the disruption of the existing G/-CD-MWCNT complex and to rearrangement in a different ADA/-CD-MWCNT/G supramolecular assembly.
Fig. 2. (A) UV–vis spectra (cell path d = 1 cm, T = 298◦ K, corrected for the scattering) of free guanine ([G] = 60 M) (a) and at different [G]//[-CD-] molar ratios: [G]/[-CD] = 1:0.25 (b), 1:0.5 (c), 1:1 (d). (B) UV–vis spectra of G (60 M) (cell path d = 1 cm, T = 298◦ K, corrected for the scattering) at different [G]//[-CD] molar ratios: [G]/[-CD] = 1:0.3 (I), 1:0.4 (II), 1:0.5 (III), 1:0.6 (IV). Inset: double reciprocal plot (R2 ∼ 0.98) for G/-CD-MWCNT species (corresponding to traces I, II and III).
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Fig. 3. UV–vis spectra (cell path d = 1 cm, T = 298◦ K, corrected for the scattering) of the G/-CD-MWCNT complex at 1:0.5 molar ratio without (trace c) and with the addition of 1-adamantanol (fresh sample, trace c ) and upon storage at r.t., for 1 month (trace c ). Spectra of G/MWCNT complex at 1:0.5 molar ratio without (trace e) and with addition of 1-adamantanol (fresh sample, trace e ).
A completely dissimilar behaviour has been observed by mixing ADA to sample containing G and native MWCNT. Fig. 3 shows that the spectral profile of G/MWCNT system (curve e) was unaffected by the addition of ADA (curve e ). Interestingly these spectra evidence again a strong hypocromicity of peculiar G bands but no shift of absorption maxima, suggesting a notable similarity with spectra relative to species at higher [G]/[-CD-] molar ratio (in the 1:0.25–1:0.4 molar ratio range). Altogether these data highlight that from 1:0.25 to 1: 0.4 molar ratio, probably G is interacting with nanoplatform by stacking, whereas at lower molar ratio (from 1:0.4) CD cavity can play a key role in the complexation process. With the aim to extend the study of interaction of a purine DNA base with -CD-MWCNT nanoplatform, we focused on an antiviral nucleoside analogue, using the 9-(2-hydroxyethoxy)methylguanine (Acyclovir), the well known cutaneous, mucosal, ophthalmic and systemic anti-HSV and anti-VZV drug, as model compound. UV–vis spectrum of free Acy (Fig. 4a curve a) in aqueous solution exhibits an absorption band centred at 252 nm with a shoulder around 272 nm, which change in the intensity and profile increasing the amounts of -CD-MWCNT (from 15 to 60 M in -CD units). Generally a hypocromicity of the band and a slight red-shift of absorption maxima have been experienced at all the [Acy]//[-CD-] investigated molar ratios, and in particular a successive increase of the absorption is observed by progressive decrease of [Acy]//[-CD-] molar ratio (Fig. 4A, curves 8b–e), reaching a maximum at ∼1:1 molar ratio. Also the
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presence of an isosbestic point suggests the complexation equilibrium between drug and drug/nanohybrid system. The capability of Acy to interact with -CD-MWCNT hybrid was also confirmed by DSC analysis (Fig. 4B). The DSC thermogram of Acy shows the characteristic endothermic peak due to the drug melting at 252 ◦ C; this broadened peak is also detectable in the thermogram of physical mixture between Acy and -CD-MWCNT (black line) prepared at the same weight ratio of the complex. In the thermogram of the complex (green line) this peak disappears, indicating that Acy is no longer crystalline, due to the formation of complex. Also, by spectophotometric titrations, reported in Figs. 2B and 4A, was possible to study the association capability of the nanoplatform towards guest molecules. Both in Figs. 2B and 4A we observe the presence of pseudo-isosbestic points. These spectroscopic evidences probably refer to the complexation equilibrium between drug and drug/nanohybrid system at different molar ratios. To give more insights on strength of the drug/nanohybrid interaction, double reciprocal plots of 1/A versus 1/[-CD-] are reported (insets of Figs. 2B and 4A; A is the difference of absorption of G or Acy respectively in the absence and presence of -CD-MWCNT). The plots exhibit a fair linearity (the linear regression coefficients R2 are ∼ =0.98 and ∼ =0.94 for titrations with G and Acy, respectively). The relative association equilibrium constant for G/-CD-MWCNT complex was estimated to be ∼ =3.2 × 104 M−1 (inset of Fig. 2B), by considering host concentration till 30 M (i.e. ≤1:0.5 molar ratio). As anticipated by inspection of Fig. 2B, at higher host concentration (i.e. >1:0.5 molar ratio), a slight increase of absorbance and a further shift were evident. This result could refer to a formation of new equilibrium species at 1:0.5 guest/host molar ratio with the involvement of CD cavity as complexation site of G. However it was not easy to discriminate binding constant for these equilibria because of the little spectral changes. In the case of Acy, we plot −1/A as an increasing function of 1/[-CD-] (inset of Fig. 4A). In the plot two phases are evident: at low molar ratios range, by increasing the amount of nanohybrid from 1:0.5 to 1:1 [Acy]/[-CD-] molar ratios (spectra a–c), we estimated an association constant around 6 × 104 M−1 . At higher molar ratios (i.e. at lower concentration of -CD-MWCNT, from 1:0.25 to 1:0.5 guest/host molar ratios), the spectral changes were not valuable to permit a reliable estimation of binding constant. Altogether these results rely with a stronger interaction of nanohybrid with Acy with respect to G and the driving force could be furnished by the additional polar chain on Acy. Significant spectral changes were registered till 1:0.5 [G]/[CD-] and 1:1 [Acy]/[-CD-] molar ratios, respectively, whereas spectrum of [G]/[-CD-] at 1:1 molar ratio was less informative.
Fig. 4. (A) UV–vis spectra of Acy (60 M, trace a) (cell path d = 1 cm, T = 298◦ K, corrected for the scattering) upon titration with -CD-MWCNT nanohybrid at different [Acy]//[-CD-] molar ratios: [Acy]/[-CD-] = 1:0.25 (b), 1:0.5 (c), 1:0.75 (d), 1:1 (e). Inset: double reciprocal plot (R2 ∼ 0.94) for Acy/-CD-MWCNT species (corresponding to c–e traces). (B) DSC thermograms of Acyclovir, Acy/-CD-MWCNT complex, Acy and -CD-MWCNT physical mixture and -CD-MWCNT.
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Fig. 5. Release profile of Acy from -CD-MWCNT in PBS at pH 7.4 and 37 ◦ C (mean ± SD). Release profile of free Acy is reported for comparison (inset).
The recognition and entrapment of Acy versus G on -CD-MWCNT are the result of different interactions, such as hydrogen bonding between the CD and the polar groups on purine derivatives, -stacking of guanine moiety on the CNT surface and/or on the triazole ring and the phenyl group. The strength of stacking vs CD interaction was difficult to discriminate because of the high heterogeneity of medium.
Fig. 6. Plaque reduction assay. The number of HSV-1 plaques was evaluated by using Vero cells in the presence of -CD-MWCNT, Acy and Acy -CD-MWCNT complexes at three different concentrations (60, 6 and 0.6 M) respectively.
using a standard plaque reduction assay. The screening has been performed in medium supplemented with 1% FBS, at three different concentrations of guest (60, 6, 0.6 M), using free Acyclovir and the nanohybrid as controls, with [-CD-] ∼ = 60, 6, 0.6 M (Fig. 6). The complex inhibits plaque formation, without affecting cell viability and morphology. Indeed, with respect to the controls, plaques number and size-morphology were significantly reduced. We found that the antiviral activity of the complex was improved with respect to the free drug, probably due to an enhanced bioavailability.
3.3. Drug delivery 4. Conclusion The drug loading capacity and drug release profile are important performance indicators of drug delivery materials. Thus, we determined the G and Acy loading capacity by preparing the G/CD-MWCNT and the Acy/-CD-MWCNT complexes at 10:1 molar ratio. Free, unbound drugs in the -CD-MWCNT solution were removed by filtration on a Millipore membrane of 0.22 m, until free, unbound drugs were no more detected. On the basis of optical absorbance data and molar extinction coefficients, we estimated that the drug loading is about 6.5% for G and 12.2% for Acy, respectively. The release experiments have been performed in PBS at pH 7.4, using the dialysis bag technique (Fig. 5). A delayed release of the drug from the platform, starting after 4 h and reaching approximately 15% after 8 days, has been registered. No initial burst effect has been observed, proving that the drug is not weakly adsorbed onto the surface. Conversely, free Acy has been found to dissolve rapidly (1 h), and a 15% of drug can be detected in the release compartment after just 24 h. The incomplete release obtained for Acy-loaded -CD-MWCNT might be related to a strong interaction with the nanoplatform sites (for 1:1 complex, kass is ∼ =6 × 104 M−1 ). The attempt of comparing Acy release kinetics from -CD-MWCNT system with release from precursor (3) failed because its lower dispersibility in aqueous medium (of about an order of magnitude) with respect to -CD-MWCNT nanohybrid (data not shown). 3.4. Plaque reduction assay The toxicity of our nanoplatform to affect Vero cells viability was studied by Trypan blue exclusion assay. The cells were exposed for 24 h to various concentrations (100, 10 g mL−1 of CD-MWCNT and Acy/-CD-MWCNT complex, which correspond to [Acy] = [-CD-] ∼ = 60 M and ∼ =6 M, respectively). A cell viability of 82% and 85% for -CDMWCNT and Acy/-CD-MWCNT respectively, was observed at the highest tested concentration (100 g mL−1 ), while no significant cell death was detected at the lower tested concentration (data not shown). Finally, the antiviral activity of Acyclovir loaded into the nanohybrid against HSV-1 in vitro was preliminarily evaluated
-Cyclodextrin/multiwalled carbon nanotubes nanohybrid as platform for the entrapment and delivery of drugs was synthesized by click chemistry approach and characterized by several complementary techniques. The drug binding abilities of nanocarrier towards guanine and modified nucleoside were proved. The association equilibrium constants for G/-CD-MWCNT and Acy/CD-MWCNT complexes were estimated to be >104 M−1 , pointing out the strong affinity of our nanoplatform towards guanine-based drugs, as the integrated result of several contributes, comprising the interaction with CD cavity and/or the -stacking on the CNT surface and/or on 1,2,3-triazole ring and phenyl group of the linker. The -CD-MWCNT was able to bind and sustain the release of Acyclovir in physiological conditions, indicating, by preliminary plaque reduction assay, an anti HSV-1 effect higher than the free drug. In agreement with these findings, -CD-MWCNT hybrid could be proposed as a promising biocompatible and bioavailable nanoplatform for the loading of various kinds of drugs. In this scenario a combination of therapeutic guests could be linked by supramolecular approach to the platform in aqueous medium according to their affinity with binding sites. Acknowledgement EuroBioSAS-OP-009 (Intelligent Cell Surface project); PRIN 20109Z2XRJ 010. Appendix A. Supplementary data Supplementary data associated with this article (MWCNT characterization, XPS, IR, FEG-SEM, TEM and AFM analyses) can be found, in the online version, at http://dx.doi.org/10.1016/j. colsurfb.2014.09.025. References [1] K.K. Jain, Exp. Opin. Drug Discov. 7 (2012) 1029–1037. [2] D. Iannazzo, A. Piperno, A. Pistone, G. Grassi, S. Galvagno, Curr. Med. Chem. 20 (2013) 1333–1354.
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-Cyclodextrin-grafted on multiwalled carbon nanotubes as versatile nanoplatform for entrapment of guanine-based drugs Daniela Iannazzo a,1 , Antonino Mazzaglia b,∗,1 , Angela Scala b,1 , Alessandro Pistone a,1 , Signorino Galvagno a , Maurizio Lanza c , Cristina Riccucci d , Gabriel Maria Ingo d , Ivana Colao e , Maria Teresa Sciortino e , Francesco Valle f , Anna Piperno g,∗∗ , Giovanni Grassi g a Dipartimento di Ingegneria Elettronica, Chimica e Ingegneria Industriale, Università di Messina, C.da di Dio, 98166 Messina, Italy b Consiglio Nazionale delle Ricerche CNR-ISMN c/o Dip. di Scienze Chimiche dell’Università di Messina, Viale Ferdinando Stagno D’Alcontres 31, 98166 Messina, Italy c Consiglio Nazionale delle Ricerche CNR-IPCF, Viale Ferdinando Stagno D’Alcontres, 98166 Messina, Italy d Consiglio Nazionale delle Ricerche CNR-ISMN, UOS Montelibretti, Via Salaria, 00015 Monterotondo Stazione, Rome, Italy e Dipartimento di Scienze Biologiche e Ambientali, Università di Messina, Viale Ferdinando Stagno D’Alcontres, 98166 Messina, Italy f Consiglio Nazionale delle Ricerche CNR-ISMN, Via Piero Gobetti 101, 40129 Bologna, Italy g Dipartimento di Scienze Chimiche, Università di Messina, Viale Ferdinando Stagno D’Alcontres 31, 98166 Messina, Italy
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Article history: Received 30 May 2014 Received in revised form 1 September 2014 Accepted 14 September 2014 Available online xxx Keywords: Click chemistry Multiwalled carbon nanotubes -Cyclodextrins Guanine Acyclovir
a b s t r a c t The design of -cyclodextrin/multiwalled carbon nanotubes hybrid (-CD-MWCNT) as nanoplatform for the entrapment and delivery of guanine based drugs is described here. The functionalized carbon nanomaterials have been characterized by XPS spectroscopy, electron microscopy (FEG-SEM and TEM), AFM, TGA, and FT-IR to achieve insights on structure, morphology and chemical composition. The drug binding abilities of nanocarrier towards the guanine (G) and Acyclovir (Acy) were proved by UV–vis and DSC experiments. Host–guest equilibrium association constants and drug loading have been evaluated for G/-CD-MWCNT and Acy/-CD-MWCNT complexes. The release studies showed a sustained delivery of Acy without initial burst effect confirming a strong interaction of drug with the nanoplatform sites. The preliminary antiviral data indicated that the Acyclovir loaded into the -CD-MWCNT platform interferes with HSV-1 replication and the antireplicative effect was higher than the free drug. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Over the last decade, functionalized carbon nanotubes (f-CNT) are emerging as promising delivery systems for the transport and release into the cells of small drugs, nucleic acids or proteins [1–4]. Chemical modifications on CNT, changing the physicochemical nature of CNT dispersions (CNT individualization against small or large bundles), allow to overcome the complete aqueous insolubility of unmodified CNT (pristine) and decrease their potential risk from medical use. To improve the CNT dispersibility in a biological
∗ Corresponding author. Tel.: +39 0903974108; fax: +39 0903974108. ∗∗ Corresponding author. Tel.: +39 090 6765173; fax: +39 090 393897. E-mail addresses: [email protected] (A. Mazzaglia), [email protected] (A. Piperno). 1 These authors contributed equally to this work.
milieu, several non-covalent and covalent chemical modification methods have been explored and there is increasing evidence that biocompatibility and biodegradation of CNT can be achieved by appropriate functionalization [5]. Moreover, the nature of the groups, linked covalently or by supramolecular approach to the CNT surface, affects the pathways of cellular internalization, i.e. passive diffusion by means of “nanoneedle” or energy-dependent cellular uptake [6]. CNT surface modification by macrocyclic host molecules, such as crown ethers [7,8], calixarenes [9], cyclodextrins [10–13], cucurbiturils [14], has gained significant attention because of their important roles in supramolecular chemistry. Among a wide range of macrocyclic molecules, -cyclodextrins (CD), cyclic oligosaccharides based on seven d-glucose units linked by ␣-1,4 glucose bond, are well established drug carriers with high biocompatibility able to host different guest molecules by inclusion into the cavity or interaction with external rims. The combination of the physical properties of CNT and the molecular recognition ability
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of -CD grafted on the CNT surface generates interesting constructs with promising applications in different fields such as environment [15], catalysis [16], chemical sensing [17]. Although different synthetic procedures have been proposed to obtain cyclodextrin grafted-CNT [15–20], at the best of our knowledge, no data are available regarding the effectiveness of -CD-CNT covalently conjugates as delivery system able to sustain the release of small drugs. Our ongoing programme is aimed for the discovery of new antiviral drugs [21–26], with enhanced therapeutic efficiency and reduced unwanted side effects. In this direction our interest was focused on the synthesis of a new nanocarrier-based delivery system for the multimodal anchorage of antiviral drugs. Here, we report an approach to fabricate -cyclodextrin/multiwalled carbon nanotubes (-CD-MWCNT) nanohybrid as prospective platform for drug delivery. The present work newly describes the synthesis of the nanohybrid by click-chemistry, characterization by complementary techniques and recognition ability of the system towards the guanine, a poor soluble DNA base. Afterwards, the interaction of the nanohybrid with Acyclovir (Acy), the prototype of acyclic nucleoside analogues, active on three human alpha herpesvirus (HSV-1, HSV-2 and VZV) has been investigated. In our study, Acy was selected as hydrophilic antiviral drug model, in virtue of the easy reliability of its biological assay and for its physical properties that cause poor oral bioavailability. For improving the efficacy of Acy treatment and decreasing its adverse side effects, many technological approaches comprising pro-drug preparations, innovative formulations and subcutaneous delivery system that provide a long-lasting sustained release have been proposed [27,28]. The release studies of Acy loaded on our nanoplatform showed a slow release, about 15% after 8 days, without the initial burst effect. The preliminary antiviral data indicated that Acy/-CD-MWCNT complex interferes with HSV-1 replication and the antireplicative effect is higher than the free drug. As proof of principle, this study has demonstrated the reliability of -cyclodextrin/multiwalled carbon nanotubes nanohybrid as attractive platform for the loading and delayed release of drugs. 2. Materials and methods 2.1. Materials MWCNT were produced by catalytic chemical vapor deposition (CCVD) from isobutane on Fe/Al2 O3 catalyst [29]; then synthesized MWCNT were subjected to purification and oxidation reaction to obtain carboxylated multiwalled carbon nanotubes (MWCNT-Ox) as previously reported [30]. All other reagents were of the highest commercial grade available and were used as received or were purified by distillation or re-crystallization when necessary. All the solutions used for spectroscopic characterization were prepared in ultrapure microfiltered water (Galenica Senese) and analysed at 298 K. 2.2. Preparation of MWCNT-alkyne (3) MWCNT-Ox (1) [30] (400 mg) were dispersed in odichlorobenzene (100 mL), sonicated for 15 min at room temperature; then a solution of p-(2-propylnyloxy)-benzamine (2) [20] (2 g dissolved in 200 mL of dry acetonitrile) was added, and the mixture was sonicated for 30 min at room temperature. The reaction mixture was stirred for 10 min under helium flow, added with 3.0 mL of isoamyl nitrite and stirred at 60 ◦ C for 24 h under helium flow. The mixture was then cooled, diluted with ethanol and filtered under vacuum on Millipore membrane of 0.1 m. The solid residue was washed with water, ethanol, chloroform
and ethyl ether (3 × 50 mL) and each time sonicated for 5 min. The residue was dried under vacuum at 50 ◦ C to give 402 mg of MWCNT-alkyne (3). From TGA analysis the amount of grafted alkyne moieties onto the surface of MWCNT could be estimated to be ∼5 wt% corresponding to ∼0.09 mmol g−1 . 2.3. Preparation of ˇ-CD-MWCNT nanohybrid (5) 100 mg of (3) were dispersed in 15 mL of dry and degassed DMF. The mixture was sonicated for 15 min under helium flow and then treated with 125 mg of mono-6-deoxy-6-azido--cyclodextrin (4) [31] (125 mg, 0.125 mmol), CuSO4 (15 mg, 0.085 mmol) and Na ascorbate (35 mg, 0.17 mmol). The reaction mixture was sonicated for 10 min and heated at 85 ◦ C for 48 h under helium flow; then cooled to room temperature, diluted with ethyl acetate (50 mL) and filtered under vacuum on Millipore membrane of 0.1 m. The solid residue was washed with water, ethanol, and ethyl ether (3 × 50 mL) and each time sonicated for 5 min. The residue was dried under vacuum at 50 ◦ C to give 70 mg of -CD-MWCNT (5). From TGA analysis the amount of -CD grafted onto the surface of MWCNT could be estimated to be ∼6 wt% corresponding to ∼0.055 mmol g−1 . 2.4. Investigation of drugs/ˇ-CD-MWCNT interaction and complexes preparation 2.4.1. Interaction of ˇ-CD-MWCNT with G A stock dispersion of nanohybrid (5) has been prepared by dispersing -CD-MWCNT (1 mg/mL) in ultrapure water and sonicating the mixture for 2 min. A stock solution of 600 M of guanine (G) in MeOH has been prepared and various aliquots of 200 L of this solution have been evaporated. Organic films of G have been re-dissolved, by increasing amounts of nanohybrid aqueous dispersions (from 15 to 60 M) at different [G]//[-CD-] molar ratios. In all the systems [G] was 60 M. 2.4.2. Interaction of ˇ-CD-MWCNT with Acy The Acy/-CD-MWCNT interaction was investigated by titrating an aqueous solution of Acy (60 M) with increasing amounts of an aqueous dispersion of -CD-MWCNT (1 mg/mL). -CD-MWCNT has been sonicated for 2 min, and added from 15 to 60 M at different [Acy]//[-CD-] molar ratios. All the systems have been stirred at 298 K for 2 days, equilibrated for 1 day at r.t. and then analysed. In all the samples the concentration of host nanohybrid is given as in -CD units [-CD-] grafted onto the surface of MWCNT, as evaluated by TGA (∼ =0.055 mmol g−1 ). The G/MWCNT complex was prepared with the same procedure used for G/-CD-MWCNT complexes. Pristine MWCNT (0.7 mg/mL) was dispersed under 2 min sonication in ultrapure water. Competition experiments were carried out by adding an excess of (∼ =20 eq) of 1-adamantanol (ADA) to the complex [G]/[-CD-] 1:0.5 molar ratio. The complex was preventively centrifuged, the supernatant was removed and the residue was re-dispersed in aqueous solution. After adding the ADA, the dispersion was mixed by vortex and analysed freshly or upon storage at room temperature. The association constants have been estimated according to the equation of Benesi and Hildebrand (double-reciprocal method) [32]. 2.5. Drug loading The G/-CD-MWCNT complex and the Acy/-CD-MWCNT complex at 10:1 molar ratio were centrifuged with 2000 r min−1 for 6 min and repeatedly filtered with a Millipore membrane of 0.22 m, until free, unbound drugs were no more detected.
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The precipitates were re-dispersed in 2 mL of ultrapure microfiltered water and UV–vis spectra were recorded. On the basis of optical absorbance data and molar extinction coefficients (ε247 ∼ = 1670 M−1 cm−1 ; ε252 ∼ = 11,400 M−1 cm−1 , for G and Acy respectively), a 6.5% and 12.2% drug loading was calculated for guanine and Acy, respectively, using the following equation: Drug loading = (drug weight in the nanoparticles/weight of the nanoparticles) × 100. 2.6. In vitro release study The in vitro release experiments were performed in 10 mM phosphate buffer containing NaCl (137 mM) and KCl (2.7 mM) at pH 7.4 (PBS) by a dialysis method. A known amount of the 1:1 complex (2.5 mg) was dispersed in 2.5 mL of PBS and placed in a dialysis bag (MWCO = 3500 Da, Spectra/Por® ). The sample was plunged in PBS of higher volume (15 mL) under stirring (sink condition) and kept at 37 ◦ C. At fixed times, 1 mL of release medium was withdrawn and replaced with an equal volume of fresh and analysed by UV–vis by using ε252 . The release of free Acy was carried out using 2.5 mL of a stock solution of Acy (1.4 mg in 100 mL PBS) in the inner bag and 15 mL of PBS in the external bag. All the experiments were carried out in triplicate. 2.7. Cytotoxicity and plaque reduction assay Vero cells were collected and cytotoxicity was evaluated by Trypan blue exclusion standard assay (Sigma–Aldrich). Percentage of death cells was evaluated in a Burker chamber using 20× magnification of an inverted microscope. The virus was diluted to yield 30 plaques/100 L. All the volumes were inoculated on monolayers of Vero cells in 24-well dishes and incubated for 1 h at 37 ◦ C. After the incubation time, the inoculum was removed and the monolayers were covered with Dulbecco’s-modified Eagle’s medium containing 0.3% methylcellulose included compounds at different concentrations (60, 6 and 0.6 M). After 3 days of incubation at 37 ◦ C, the medium was aspirated and the cells were then fixed, stained with crystal violet, and visualized at 10× magnification with an inverted microscope for plaque detection. The assays were performed in duplicate for each dilution. 3. Results and discussion 3.1. Synthesis and characterization of ˇ-CD-MWCNT As substrate for the synthesis of -CD-MWCNT nanohybrid (Fig. 1), we have selected the carboxylated multiwalled carbon nanotubes (MWCNT-Ox), which did not show cytotoxicity in our previous studies [30,33]. MWCNT-Ox obtained by oxidation of pristine MWCNT contains 1.8 mmol g−1 of free OH groups of which about 1.2 mmol g−1 are carboxylic group available for the chemical derivatization [30,33]. Our approach relies on the derivatization of MWCNT-Ox (1) with p-(2-propylnyloxy)benzammine (2), using diazotization-coupling procedure, to obtain (3). Alkyne-functionalized MWCNT (3) has been successfully coupled by click reaction with the monoazide--CD (4) (-CD-N3 ), giving the -CD-MWCNT nanohybrid (5), through Cu-mediated acetylene-azide coupling (CuAAC) reaction (Huisgen cycloaddition) using CuSO4 /Na-ascorbate as metal source. The functionalized carbon nanomaterials have been characterized by XPS, microscopy (FEG-SEM, TEM, AFM), TGA and FT-IR to gain insights on structure, morphology and chemical composition. XPS spectra of pristine MWCNT showed only an intense C 1s peak [34] at a binding energy ca. 284 eV, while for the all functionalized CNT, the detection of a N 1s peak at ca. 400 eV and O 1s peak at ca. 532 eV indicated the success of the functionalization processes (see Fig. 1 SM). According to
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our previous results [35], MWCNT-Ox XPS spectra show the presence of a N 1s peak at ca. 400 eV indicating that the oxidative process is not selective towards the formation of carboxylic groups, but also some other nitrogenated chemical groups are introduced. XPS data provide an estimate of the functionalization degree (m), via the comparison of the amount of nitrogen and carbon (N:C ratio), and oxygen and carbon (O:C ratio), in agreement with the m calculated by TGA. The m of MWCNT-Ox (1), MWCNT-alkyne (3) and -CD-MWCNT (5) was deduced comparing the TGA data obtained by thermal decomposition of the surface groups heated up to 700 ◦ C under inert atmosphere and was found of 18%, 23% and 29% respectively (Fig. 1). According to literature data [36,37], the TGA curve for -CD-MWCNT (5) exhibits two steps of weight loss, contributed from the loss of residual water (in the range 30–190 ◦ C) and the loss of -CD in the range of 200–450 ◦ C, as shown by DTG curve (inset of Fig. 1). Thus, the amount of -CD grafted onto the surface of MWCNT could be estimated to be ∼6 wt% corresponding to ∼0.055 mmol g−1 . The FT-IR spectra (see Fig. 2 SM) indicated the successful covalent grafting of the -CD moiety on the MWCNT. For oxidized MWCNT (1) the curve shows the peaks at 3445 (O H vibration), 2930 (C H stretching), 1710 and 1150 (C O and C O stretching), 1630 (C C stretching) cm−1 . After diazotization-coupling, the IR spectrum of alkyne modified MWNT (3) exhibits the characteristic absorption of alkyne group at 2130 cm−1 . The characteristic peaks due to the strong absorptions of C O stretching vibration of -CD at 1220, 1170, 1100 and 1030 cm−1 can be clearly seen in the IR spectrum of the nanohybrid (5). FEG-SEM analyses showed a high degree of bundling for MWCNT-Ox (1), while in -CD-MWCNT (5) carbon nanotubes appear entangled together inside a smooth matrix. This topographic aspect can be explained by partial cross-linking of the carbon nanotubes and the -cyclodextrin with this latter homogeneously deposited along the nanotube shaping densely packed carbon nanotubes (see Fig. 3 SM). TEM images of pristine MWCNT showed a clear multi-walled tube structure with a diameter close to 15–20 nm [34]. After the acid treatment, MWCNT-Ox (1) are dispersed and shortened on account of the severe oxidation of nitric–sulphuric acid mixture (see Fig. 4a SM). The external walls of MWCNT-Ox appear severely eroded with several continuous graphite layers often truncated and significant faults between graphite carbon planes; no amorphous layers on the external walls of MWCNT-Ox were observed (see Fig. 4b SM). TEM analyses confirmed the success of the grafting of cyclodextrin to carbon nanotubes; the -cyclodextrin molecules densely decorate the carbon nanotubes (see Fig. 4c SM) and a diffuse hazy contrast on the external walls (indicated by arrow), due to linkage with amorphous -cyclodextrin moieties, was observed (see Fig. 4d SM). AFM images (see Fig. 5a–c SM) have allowed to characterize the morphology of the nanotubes both bare and functionalized. It is noteworthy that when dealing with nanometer sized objects the parameter that is less affected by the tip convolution is the height, because the lateral size is usually enlarged and influenced because of the tip shape. The functionalization of MWCNT with -CD leads to an increase in the lateral nanotube size that is particularly well appreciated in the section analysis where a net height increase is evident. -CD-MWCNT nanohybrid exhibits a multiwalled tube structure of 15–20 layers with an average length of 200–1000 nm. The height was found to be about 5 nm, as determined by AFM analysis (see Fig. 5c SM). 3.2. Complexation capability towards guanine-based drugs In order to probe the recognition ability of -CD-MWCNT nanohybrid, guanine (G), a poor soluble DNA base, and Acyclovir, an
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Fig. 1. Right: preparation of -CD-MWCNT nanohybrid (5) with the values of m% 700 ◦ C, N/C ratio and O/C ratio obtained from TGA and XPS data. Left: TGA profile of MWCNT and 1, 3 and 5; in the inset DTG curve of 5.
acyclic analogue of the natural nucleoside 2 -deoxyguanine, were selected as molecular models in the complexation experiments. Studies concerning the DNA bases interactions with cyclodextrin modified carbon nanotubes are rare, except for few reports [17,38], which suggest that purine bases have a strong affinity towards the CD cavity. In our study, the complexation capability of -CD-MWCNT towards G was investigated by UV–vis. Fig. 2A shows the UV–vis spectra of G in ultrapure water containing various amounts of -CD-MWCNT (from 1:0.25 to 1:1 [G]/[-CD-] molar ratio). It is clear that the UV–vis spectrum of G changes with the addition of -CD-MWCNT. In particular, by increasing the amount of -CD-MWCNT, at the highest molar ratio (1:0.25), the UV–vis spectrum (Fig. 2A, curve b) shows hypocromicity of the G bands centred at 247 and 278 nm, presumably due to G present in two different environments, ascribable to free G in equilibrium with G allocated on -CD-MWCNT. The propensity of purine base to interact with nanohybrid is more evident at lower molar ratio (1:0.5; Fig. 2A, curve c), as shown by changes of the peculiar double band whose maxima were red-shifted from 247 to 253 nm and blueshifted from 278 to 273 nm, respectively. At 1:1 molar ratio (Fig. 2A, curve d), only a wide band (with a maximum at 270 nm and a shoulder at 256 nm) appears and this spectral change was tentatively ascribed to a strong interaction of G with the nanohybrid. The modifies in the UV–vis spectra can plausibly depend on the several interactions of G with the different platform sites, comprising the selective inclusion into the CD cavity and the stacking [12] on the CNT surface and/or on 1,2,3-triazole ring and phenyl
group of the linker. With the aim to characterize G/nanohybrid species, we underlined the spectral changes of G by increasing the amount of nanohybrid, in a selected range of [G]//[-CD-] molar ratios (from 1:0.3 to 1:0.6, see Fig. 2B). As anticipated, no shift of absorption maxima has been observed till about 1:0.4 molar ratio. On the other hand at lower molar ratios (from 1:0.5 molar ratio), evident wavelength shifts have been observed and a slight recovery of absorption intensity. In detail, the presence of a nearly-isosbestic point between 24 and 36 M in -CD units, implies an equilibrium between different species. To elucidate the prominent interaction sites of G and investigate the role of the CD cavity in the complexation process, 1-adamantanol (ADA) has been chosen as a competitive guest for G for its ability to strongly interact into the inner cavity of CD, not possessing bands in the absorption region of DNA base. Fig. 3 shows the UV–vis spectra of G/-CD-MWCNT complex (prepared at 1:0.5 [G]/[-CD-] molar ratio, curve c) after addition of ADA. Spectra of ADA/-CD-MWCNT/G were registered on fresh sample after few minutes, and after 1 month (curves c and c , respectively). We hypothesize an ADA involvement in the complexation equilibrium, probably moving G from the hydrophobic cavity of the CD to different recognition sites i.e. CNT surface and/or to the triazole and phenyl groups. The UV–vis spectra registered after 1 month clearly indicate a noticeable change referable to the disruption of the existing G/-CD-MWCNT complex and to rearrangement in a different ADA/-CD-MWCNT/G supramolecular assembly.
Fig. 2. (A) UV–vis spectra (cell path d = 1 cm, T = 298◦ K, corrected for the scattering) of free guanine ([G] = 60 M) (a) and at different [G]//[-CD-] molar ratios: [G]/[-CD] = 1:0.25 (b), 1:0.5 (c), 1:1 (d). (B) UV–vis spectra of G (60 M) (cell path d = 1 cm, T = 298◦ K, corrected for the scattering) at different [G]//[-CD] molar ratios: [G]/[-CD] = 1:0.3 (I), 1:0.4 (II), 1:0.5 (III), 1:0.6 (IV). Inset: double reciprocal plot (R2 ∼ 0.98) for G/-CD-MWCNT species (corresponding to traces I, II and III).
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Fig. 3. UV–vis spectra (cell path d = 1 cm, T = 298◦ K, corrected for the scattering) of the G/-CD-MWCNT complex at 1:0.5 molar ratio without (trace c) and with the addition of 1-adamantanol (fresh sample, trace c ) and upon storage at r.t., for 1 month (trace c ). Spectra of G/MWCNT complex at 1:0.5 molar ratio without (trace e) and with addition of 1-adamantanol (fresh sample, trace e ).
A completely dissimilar behaviour has been observed by mixing ADA to sample containing G and native MWCNT. Fig. 3 shows that the spectral profile of G/MWCNT system (curve e) was unaffected by the addition of ADA (curve e ). Interestingly these spectra evidence again a strong hypocromicity of peculiar G bands but no shift of absorption maxima, suggesting a notable similarity with spectra relative to species at higher [G]/[-CD-] molar ratio (in the 1:0.25–1:0.4 molar ratio range). Altogether these data highlight that from 1:0.25 to 1: 0.4 molar ratio, probably G is interacting with nanoplatform by stacking, whereas at lower molar ratio (from 1:0.4) CD cavity can play a key role in the complexation process. With the aim to extend the study of interaction of a purine DNA base with -CD-MWCNT nanoplatform, we focused on an antiviral nucleoside analogue, using the 9-(2-hydroxyethoxy)methylguanine (Acyclovir), the well known cutaneous, mucosal, ophthalmic and systemic anti-HSV and anti-VZV drug, as model compound. UV–vis spectrum of free Acy (Fig. 4a curve a) in aqueous solution exhibits an absorption band centred at 252 nm with a shoulder around 272 nm, which change in the intensity and profile increasing the amounts of -CD-MWCNT (from 15 to 60 M in -CD units). Generally a hypocromicity of the band and a slight red-shift of absorption maxima have been experienced at all the [Acy]//[-CD-] investigated molar ratios, and in particular a successive increase of the absorption is observed by progressive decrease of [Acy]//[-CD-] molar ratio (Fig. 4A, curves 8b–e), reaching a maximum at ∼1:1 molar ratio. Also the
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presence of an isosbestic point suggests the complexation equilibrium between drug and drug/nanohybrid system. The capability of Acy to interact with -CD-MWCNT hybrid was also confirmed by DSC analysis (Fig. 4B). The DSC thermogram of Acy shows the characteristic endothermic peak due to the drug melting at 252 ◦ C; this broadened peak is also detectable in the thermogram of physical mixture between Acy and -CD-MWCNT (black line) prepared at the same weight ratio of the complex. In the thermogram of the complex (green line) this peak disappears, indicating that Acy is no longer crystalline, due to the formation of complex. Also, by spectophotometric titrations, reported in Figs. 2B and 4A, was possible to study the association capability of the nanoplatform towards guest molecules. Both in Figs. 2B and 4A we observe the presence of pseudo-isosbestic points. These spectroscopic evidences probably refer to the complexation equilibrium between drug and drug/nanohybrid system at different molar ratios. To give more insights on strength of the drug/nanohybrid interaction, double reciprocal plots of 1/A versus 1/[-CD-] are reported (insets of Figs. 2B and 4A; A is the difference of absorption of G or Acy respectively in the absence and presence of -CD-MWCNT). The plots exhibit a fair linearity (the linear regression coefficients R2 are ∼ =0.98 and ∼ =0.94 for titrations with G and Acy, respectively). The relative association equilibrium constant for G/-CD-MWCNT complex was estimated to be ∼ =3.2 × 104 M−1 (inset of Fig. 2B), by considering host concentration till 30 M (i.e. ≤1:0.5 molar ratio). As anticipated by inspection of Fig. 2B, at higher host concentration (i.e. >1:0.5 molar ratio), a slight increase of absorbance and a further shift were evident. This result could refer to a formation of new equilibrium species at 1:0.5 guest/host molar ratio with the involvement of CD cavity as complexation site of G. However it was not easy to discriminate binding constant for these equilibria because of the little spectral changes. In the case of Acy, we plot −1/A as an increasing function of 1/[-CD-] (inset of Fig. 4A). In the plot two phases are evident: at low molar ratios range, by increasing the amount of nanohybrid from 1:0.5 to 1:1 [Acy]/[-CD-] molar ratios (spectra a–c), we estimated an association constant around 6 × 104 M−1 . At higher molar ratios (i.e. at lower concentration of -CD-MWCNT, from 1:0.25 to 1:0.5 guest/host molar ratios), the spectral changes were not valuable to permit a reliable estimation of binding constant. Altogether these results rely with a stronger interaction of nanohybrid with Acy with respect to G and the driving force could be furnished by the additional polar chain on Acy. Significant spectral changes were registered till 1:0.5 [G]/[CD-] and 1:1 [Acy]/[-CD-] molar ratios, respectively, whereas spectrum of [G]/[-CD-] at 1:1 molar ratio was less informative.
Fig. 4. (A) UV–vis spectra of Acy (60 M, trace a) (cell path d = 1 cm, T = 298◦ K, corrected for the scattering) upon titration with -CD-MWCNT nanohybrid at different [Acy]//[-CD-] molar ratios: [Acy]/[-CD-] = 1:0.25 (b), 1:0.5 (c), 1:0.75 (d), 1:1 (e). Inset: double reciprocal plot (R2 ∼ 0.94) for Acy/-CD-MWCNT species (corresponding to c–e traces). (B) DSC thermograms of Acyclovir, Acy/-CD-MWCNT complex, Acy and -CD-MWCNT physical mixture and -CD-MWCNT.
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Fig. 5. Release profile of Acy from -CD-MWCNT in PBS at pH 7.4 and 37 ◦ C (mean ± SD). Release profile of free Acy is reported for comparison (inset).
The recognition and entrapment of Acy versus G on -CD-MWCNT are the result of different interactions, such as hydrogen bonding between the CD and the polar groups on purine derivatives, -stacking of guanine moiety on the CNT surface and/or on the triazole ring and the phenyl group. The strength of stacking vs CD interaction was difficult to discriminate because of the high heterogeneity of medium.
Fig. 6. Plaque reduction assay. The number of HSV-1 plaques was evaluated by using Vero cells in the presence of -CD-MWCNT, Acy and Acy -CD-MWCNT complexes at three different concentrations (60, 6 and 0.6 M) respectively.
using a standard plaque reduction assay. The screening has been performed in medium supplemented with 1% FBS, at three different concentrations of guest (60, 6, 0.6 M), using free Acyclovir and the nanohybrid as controls, with [-CD-] ∼ = 60, 6, 0.6 M (Fig. 6). The complex inhibits plaque formation, without affecting cell viability and morphology. Indeed, with respect to the controls, plaques number and size-morphology were significantly reduced. We found that the antiviral activity of the complex was improved with respect to the free drug, probably due to an enhanced bioavailability.
3.3. Drug delivery 4. Conclusion The drug loading capacity and drug release profile are important performance indicators of drug delivery materials. Thus, we determined the G and Acy loading capacity by preparing the G/CD-MWCNT and the Acy/-CD-MWCNT complexes at 10:1 molar ratio. Free, unbound drugs in the -CD-MWCNT solution were removed by filtration on a Millipore membrane of 0.22 m, until free, unbound drugs were no more detected. On the basis of optical absorbance data and molar extinction coefficients, we estimated that the drug loading is about 6.5% for G and 12.2% for Acy, respectively. The release experiments have been performed in PBS at pH 7.4, using the dialysis bag technique (Fig. 5). A delayed release of the drug from the platform, starting after 4 h and reaching approximately 15% after 8 days, has been registered. No initial burst effect has been observed, proving that the drug is not weakly adsorbed onto the surface. Conversely, free Acy has been found to dissolve rapidly (1 h), and a 15% of drug can be detected in the release compartment after just 24 h. The incomplete release obtained for Acy-loaded -CD-MWCNT might be related to a strong interaction with the nanoplatform sites (for 1:1 complex, kass is ∼ =6 × 104 M−1 ). The attempt of comparing Acy release kinetics from -CD-MWCNT system with release from precursor (3) failed because its lower dispersibility in aqueous medium (of about an order of magnitude) with respect to -CD-MWCNT nanohybrid (data not shown). 3.4. Plaque reduction assay The toxicity of our nanoplatform to affect Vero cells viability was studied by Trypan blue exclusion assay. The cells were exposed for 24 h to various concentrations (100, 10 g mL−1 of CD-MWCNT and Acy/-CD-MWCNT complex, which correspond to [Acy] = [-CD-] ∼ = 60 M and ∼ =6 M, respectively). A cell viability of 82% and 85% for -CDMWCNT and Acy/-CD-MWCNT respectively, was observed at the highest tested concentration (100 g mL−1 ), while no significant cell death was detected at the lower tested concentration (data not shown). Finally, the antiviral activity of Acyclovir loaded into the nanohybrid against HSV-1 in vitro was preliminarily evaluated
-Cyclodextrin/multiwalled carbon nanotubes nanohybrid as platform for the entrapment and delivery of drugs was synthesized by click chemistry approach and characterized by several complementary techniques. The drug binding abilities of nanocarrier towards guanine and modified nucleoside were proved. The association equilibrium constants for G/-CD-MWCNT and Acy/CD-MWCNT complexes were estimated to be >104 M−1 , pointing out the strong affinity of our nanoplatform towards guanine-based drugs, as the integrated result of several contributes, comprising the interaction with CD cavity and/or the -stacking on the CNT surface and/or on 1,2,3-triazole ring and phenyl group of the linker. The -CD-MWCNT was able to bind and sustain the release of Acyclovir in physiological conditions, indicating, by preliminary plaque reduction assay, an anti HSV-1 effect higher than the free drug. In agreement with these findings, -CD-MWCNT hybrid could be proposed as a promising biocompatible and bioavailable nanoplatform for the loading of various kinds of drugs. In this scenario a combination of therapeutic guests could be linked by supramolecular approach to the platform in aqueous medium according to their affinity with binding sites. Acknowledgement EuroBioSAS-OP-009 (Intelligent Cell Surface project); PRIN 20109Z2XRJ 010. Appendix A. Supplementary data Supplementary data associated with this article (MWCNT characterization, XPS, IR, FEG-SEM, TEM and AFM analyses) can be found, in the online version, at http://dx.doi.org/10.1016/j. colsurfb.2014.09.025. References [1] K.K. Jain, Exp. Opin. Drug Discov. 7 (2012) 1029–1037. [2] D. Iannazzo, A. Piperno, A. Pistone, G. Grassi, S. Galvagno, Curr. Med. Chem. 20 (2013) 1333–1354.
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Please cite this article in press as: D. Iannazzo, et al., -Cyclodextrin-grafted on multiwalled carbon nanotubes as versatile nanoplatform for entrapment of guanine-based drugs, Colloids Surf. B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.09.025
