Biotin-Containing Reduced Graphene Oxide-Based Nanosystem as a

Page 1 of 30

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

Biomacromolecules

Biotin-containing Reduced Graphene Oxide-based Nanosystem as a Multi-effect Anticancer Agent: Combining Hyperthermia with Targeted Chemotherapy Nicolò Mauroa, Cinzia Scialabbaa, Gennara Cavallaroa, Mariano Licciardiab, Gaetano Giammonaab* a

Laboratory of Biocompatible Polymers, Department of “Scienze e Tecnologie Biologiche, Chimiche e

Farmaceutiche” (STEBICEF), University of Palermo, Via Archirafi, 32 90123 Palermo, Italy. b

Mediterranean Center for Human Advanced Biotechnologies (Med-Chab), Viale delle Scienze Ed.18,

90128 Palermo, Italy. KEYWORDS Graphene, targeted therapy, polymer-drug conjugates, prodrugs, doxorubicin, inulin ABSTRACT Among the relevant properties of graphene derivatives, their ability of acting as energy-converting device so as to produce heating (i.e. thermoablation and hyperthermia) was more recently took into account for the treatment of solid tumors. In this pioneering study for the first time, the in vitro RGO-induced hyperthermia was assessed and combined with the stimuli-sensitive anticancer effect of a biotinylated inulin-doxorubicin conjugate (CJ-PEGBT), hence getting to a nanosystem endowed with synergic anticancer effects and high specificity. CJ-PEGBT was synthesized by linking pentynoic acid and citraconic acid to inulin. The citraconylamide pendants, used as pH reversible spacer, was exploited to further conjugate doxorubicin, whereas the alkyne moiety was orthogonally functionalized with an azido PEG-biotin derivative by copper (II) catalyzed 1-3,dipolar cycloaddition. DSC measures, AFM and UV

ACS Paragon Plus Environment

Biomacromolecules

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

Page 2 of 30

spectrophotometry were employed to systematically investigate adsorption of CJ-PEGBT onto RGO and its physicochemical stability in aqueous media, demonstrating that a stable π-staked nanosystem can be obtained. In vitro tests using cancer breast cells (MCF-7) showed the ability of the RGO/CJ-PEGBT of efficiently killing cancer cells both via a selective laser beam thermoablation and hyperthermia-triggered chemotherapy. If compared with the non-biotinylated nanosystem, including virgin RGO and the free conjugate, RGO/CJ-PEGBT is endowed with a smart combination of properties which warrant potential as an anticancer nanomedicine.

INTRODUCTION Smart “nanomedicines” for targeted anticancer delivery are conceived to be accumulated in the tumor site in order to release their drug payload nearby the site of action, increasing its bioavailability and thus its effectiveness.1,2 Despite its great promise, the administration of nanomedicines which act with only one therapeutic mechanism might fail, provoking drug resistance phenomena.3 Hence, a multi-effect therapeutic approach which attempts to kill cancer cells through different ways is desired. The means of achieving this is not trivial, since a careful evaluation of combined therapeutic effects must be done to destroy both circulating neoplastic cells and the drug resistant one, though circumventing side toxic effects, thus avoiding recidivisms. Among the highly localized, selective and effective possible anticancer treatments, hyperthermia is the most powerful therapeutic chance to physically eradicate a tumor mass. In the field of cancer therapy, hyperthermia is a local rise in temperature of a tumor mass, usually to 40–45 °C, which can prompt immediate cellular death or hypersensitization of cancer cells towards xenobiotics.4,5 The wide interest in hyperthermia is also due to the higher thermosensitivity showed by cancer cells, mainly due to the nutrient-deprived and acidic conditions in which they reside.6 On the whole, the selective hyperthermia-triggered chemosensitization, observed in the range of 40.5– 43°C,7 provides the key to synergically combine hyperthermia with chemotherapy. However, to our knowledge it is not clear whether receiving chemotherapy plus hyperthermia is more effective than


Page 3 of 30

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

receiving

Biomacromolecules

chemotherapy

alone

in

treating

tumors.

For

instance,

whilst

thermos-sensitive

liposomes/ThermoDox® showed excellent synergic anticancer activity in vitro and in in vivo, when coupled with radiofrequency thermal ablation treatment the survival probability during the follow-up did not significantly improve.8 Though one may assume that the administration of a nanosystem which act both as hyperthermia and smart drug delivery system may aid to improve the therapeutic performance of such implements. Graphene oxide (GO) has been more recently proposed as an hyperthermia agent for anticancer therapies, demonstrating that the application of a cold laser beam can preferentially induce cell necrosis killing cancer cells.9 GO and their derivatives, such as reduced graphene oxide (RGO), have been also employed for their unique performance as drug delivery systems derived from their two-dimensional size (i.e., enormous surface available for drug adsorption and cell-carrier interactions).10-12 However, in spite of the complexity of the design proposed in such studies, which includes antibody conjugation and several tedious chemical modifications, the in vitro cytotoxic effect obtained never went beyond 70 % if compared with the control.13 Actually, the development of a nanomedicine based on RGO, providing a multi-effect therapeutic approach to kill cancer cells through a synergic action of hyperthermia and chemotherapy, requires the combination of RGO with a polymeric carrier able to efficiently load the anticancer drug and simultaneously bring the targeting agent. At this regard, inulin was chosen as candidate in this study. Inulin is a natural, biocompatible, bioeliminable and biodegradable14 polysaccharide consisting of glucopyranose end-capped (β-1,2) fructose units carrying high amount of hydroxyl functional groups regularly arranged in the polymer chain. Being highly functionalized, it is water soluble and may react with a huge array of organic compounds under eco-friendly conditions so as to confer tailor made properties useful in different biological and biomedical field.15-18 More recently, we have considered an environment-sensitive inulin-doxorubicin conjugate as anticancer agent in vitro, showing that an acceptable selectivity of its cytotoxicity towards cancer cells can be obtained by simply design polymers


Biomacromolecules

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

Page 4 of 30

endowed with high pH-sensitivity together with self-assembling ability.17 This peculiar “marriage” led us to investigate the potential of such copolymer as anticancer prodrug following biotinylation, used as targeting agent,19-22 so as to evaluate cell targeting effect in combination with RGO-induced hyperthermia. We demonstrated the biotinylation dependence of the conjugate, henceforth named CJ-PEGBT, efficacy by investigating its cytotoxicity on MCF-7 cell line in comparison with the parent conjugate, named CJ. Then we evaluated the feasibility of the adsorption of CJ and CJ-PEGBT onto RGO through differential scanning calorimetry, leading to nanosystems stable in aqueous media. Finally we tested the anticancer effects, that is chemotherapic and hyperthermic one, by evaluating the cytotoxic effect of the unbiotinilated and biotinylated nanosystems, from now on named RGO/CJ and RGO/CJ-PEGBT respectively, on MCF-7 cells also under the effect of a surgical 810 nm laser. The aim of this paper is to report on these issues.

MATERIALS AND METHODS Materials. Inulin-(2-aminoethyl)-carbamate (INU-EDA), used as starting copolymer, INU-EDA-P,C and INU-EDA-P,C-Doxo (CJ) were obtained as previously described.15,17 Doxorubicin hydrochloride (DOXO-HCl),

N-hydroxysuccinimide

(NHS),

1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide

hydrochloride (EDC-HCl), citraconic anhydride, 4-pentynoic acid (P), copper (II) sulfate pentahydrate, ascorbic acid (99%), anhydrous THF and N-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethyl]hexahydro-2oxo-(3aS,4S,6aR)-1H-thieno[3,4-d]imidazole-4-pentanamide (N3-PEG-BT) were purchased from Sigma Aldrich. The reagents were of analytic grade and used as received. Carboxyl graphene (RGO) was purchased from ACS Material (USA) and used after purification by dialysis tube with nominal molecular weight cut off 106 Da. SpectraPor dialysis tubing was purchased from Spectrum Laboratories, Inc. (Italy). 1

H NMR spectra were recorded using a Bruker Avance II 300 spectrometer operating at 300.12 MHz.


Page 5 of 30

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

Biomacromolecules

Size exclusion chromatography (SEC) was carried out using Tosho Bioscience TSK-Gel G4000 PWXL and G3000 PWXL columns connected to a Water 2410 refractive index detector. The mobile phase was a 0.1 M Tris buffer pH 8.10± 0.05 with 0.2 M sodium chloride. The flow rate was 0.6 mL min−1 and sample concentration 2.5 mg mL-1 and PEG standards (200-0.19 kDa, Polymer Laboratories Inc., USA) were used to set up calibration curve (R2 = 0.9967). Human breast cancer MCF-7 cells (purchased from Istituto Zooprofilattico Sperimentale della Lombardia e dell’ Emilia Romagna, Italy) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% v/v Fetal Bovine Serum (FBS), 2 mM L-glutamine, 100 units/mL penicillin G, 100 µg/mL streptomycin at 37°C and 5% CO2. Human venous plasma was obtained from healthy voluntary donors and immediately used. This sample was collected from the first co-author with his informed consent. Synthesis of INU-EDA-P-PEGBT,C-Doxo (CJ-PEGBT). To a solution of INU-EDA-P,C (150 mg, ≈ 0.82 mmol of repeating units) in milliQ water/DMF 6:4 (14 mL), in turn NHS (16.0 mg, 0.139 mmol), EDC-HCl (26 mg, 0.139 mmol) and doxorubicin hyodrochloride (80 mg, 0.139 mmol) were added under vigorous stirring. Then, the pH of the reaction was adjusted to 6.5-6.8 with 0.1 M NaOH and the reaction kept under these conditions until stable pH values were riched (≈ 2 h). After that, N3-PEG-BT (18.3 mg, 0.041 mmol) and copper (II) sulfate pentahydrate (1.5 mg, 0.006 mmol) were added to the reaction and the resulting solution was placed under reduced vacuum and nitrogen atmosphere three times in order to obtain an oxygen free solution. A degassed solution of ascorbic acid (5 mg / 500 µL) was added under nitrogen atmosphere and the reaction was kept under these conditions for 15 h at room temperature (Min 15 °C, Max 21 °C). Finally it was treated with a cation exchanging resin (DowexTM), filtered and dialyzed through a membrane tubing with MWCO 1 kDa and the pure product retrieved as a deep red solid. Yield 106 mg. 1H NMR 300 MHz, D2O: δ 1.78 (br, 3Hdoxo, CH3CH2O sugar ring), 1.90-2.05 (br, 3Hcitraconate,

-

OOCCH2CH3 and NHCOCH2CH3), 2.35 (br, 4Halkyl, CH2CH2C=CN), 2.50-2.60 (br, 4HBT SCH2 and


Biomacromolecules

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

Page 6 of 30

NHCOCH2) 3.00-3.50 (m, 4HEDA, NCH2CH2N), 3.62 (m, 12HPEG, CH2O and CH2N), 3.50-4.50 (7 HINU, CH2-OH; -CH-CH2-OH; -C-CH2-O-; -C-CH-OH; -CH-OH), 5.47 (1Hdoxo, O-CH-O anomeric sugar ring ), 5.52 (s, 1Hcitraconate, CHCOO-), 5.83 (s, 1Hcitraconate, CHCONH). Determination of the drug payload. The amount of doxorubicin linked in CJ and CJ-PEGBT was determined spectrophotometrically, measuring the absorbance of the sample at 480 nm, and comparing these values to that of a calibration curve obtained from standard solutions of doxorubicin hydrochloride in 0.1 M phosphate buffer at pH 7.4 (concentration range from 0.001 to 1 mg mL-1; R2 = 0.999). For comparative purposes, the content of doxorubicin was expressed as the amount of loaded doxorubicin hydrochloride per unit mass of polymer, and resulted to be 18.45 ± 0.6 % and 13.9 ± 0.2 % (w/w) for CJ and CJ-PEGBT respectively. Preparation of the RGO/Conjugate nanosystems (RGO/CJ and RGO/CJ-PEGBT). A solution of single palate RGO in water (7.4 mg mL-1) was diluted with milliQ water and the pH was adjusted to pH 7.2 ± 0.2 using 1 M sodium hydroxide. The solution so obtained had a RGO concentration of 1 mg mL-1. To a sample of this solution (1 mL) either CJ or CJ-PEGBT was added with stirring so as to get to solutions with RGO/conjugate ratio varying from 1:1 to 1:5 on a weight basis. The resultant mixture was sonicated for 15 minutes at 40 °C and then stirred at 25 °C for 2 h and stored at 4 °C over night before use. Differential scanning calorimetry of the RGO/CJ and RGO/CJ-PEGBT. The ability of both CJ and CJ-PEGBT conjugates to interact with RGO was assessed by differential scanning calorimetry (DSC), using a DSC 131 EVO (by SETARAM Instuments). Each DSC measure was performed under nitrogen atmosphere (flow 1 mL min-1) using about 2 mg of dried sample placed into an aluminum crucible. The heating rate applied was: 40 – 350 °C, 10 °C min-1. Rheological measurements. The interaction of CJ-PEGBT with RGO platelets were also investigated performing rheological tests using an DH-R2 stress controlled rheometer (TA Instruments). All tests were carried out at 25 °C using a concentric cylinders geometry (d=30.36 mm). The conformational


Page 7 of 30

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

Biomacromolecules

equilibrium study was assessed in PBS pH 7.4 and at concentration of 0.1 mg mL-1 by applying a preshear of 0.1 rad sec-1 for 10 sec followed by a flow ramp from 0.1 to 40 rad sec-1 at constant normal force. To prevent dehydration during rheological measurements, a solvent trap was placed at the top of the geometry. Each measurement was performed at least on three different sample freshly prepared. The viscosity of CJ-PEGBT, RGO and the RGO/CJ-PEGBT nanosystem at Newtonian regimen was measured in PBS pH 7.4 at the same concentration (0.1 mg mL-1) by a peak hold experiment carried out at 25 °C for 20 min and using a shear rate of 10 rad sec-1. Atomic force microscopy (AFM) analysis. The morphology of the RGO/CJ-PEGBT nanosystem and his polydispersity were determined by atomic force microscopy (AFM). AFM measurements were performed in Tapping mode in air by a Bruker Dimension FastScan microscope equipped with closedloop scanners. We used triangular FastScan A probes (resonance frequency=1400 KHz, Tip radius=5 nm). Either RGO or RGO/CJ-PEGBT platelets in a monolayer water dispersion (0.001 mg mL-1) were dropped onto a freshly cleaved mica surface and dried overnight before observation. Drug release study. For drug release studies in artificial medium, either CJ, CJ-PEGBT, RGO/CJ or RGO/CJ-PEGBT (3.0 mg) was dissolved in PBS at pH 7.4 (5 mL) and placed into a dialysis tubing with a MWCO 2 kDa. It was then immersed into PBS at pH 7.4 (40 mL) and incubated at 37°C under continuous stirring (100 rpm) in a Benchtop 808C Incubator Orbital Shaker model 420, for 48 h. Aliquots of the external medium (1 mL) were withdrawn from the outside of the dialysis tubing at scheduled time intervals and replaced with equal amount of fresh medium. The amount of doxorubicin released was evaluated spectrophotometrycally evaluating the absorbance of each sample at λ 480nm and using the ε obtained from standard solutions of doxorubicin hydrochloride in the same medium. Then, the cumulative release was determined as a function of incubation time. A parallel analysis was carried out using the same procedure above described but replacing PBS pH 7.4 with PBS at pH 5.5 after 12. All release data were compared with the diffusion profile of doxorubicin hydrochloride alone (0.5 mg) obtained by using


Biomacromolecules

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

Page 8 of 30

the same procedure. Data were corrected taking in account the dilution procedure. Each experiment was carried out in triplicate and the results were in agreement within ±5% standard error The hyperthermia-triggered drug release was evaluated treating a suspension of RGO/CJ-PEGBT with a 810nm laser (power fitted at 7x10-2 W mm-3) for 300 seconds and comparing the release obtained after an incubation time of 4h in PBS pH 7.4 with the untreated control. Evaluation of the hyperthermic effect of the nanosystem. A dispersion of RGO/CJ-PEGBT (1 : 1 w/w) in water, at concentration of 1 mg mL-1, was prepared using the protocol above described. Then, after serial dilutions of this dispersion, samples with concentration ranging from 0.05 to 1 mg mL-1 were prepared. The dispersions were treated with a 810 nm surgical diode laser (GBoxTM 15A/B by GIGA Laser) with the power fitted at 2.8x10-3 W mm-3. At fixed intervals the temperature of the dispersion was recorded and reported as a function of the exposure time. Cytotoxicity assay. The cytotoxicity of the conjugates and the mutually related systems was assessed by the MTS assay on human breast cancer (MCF-7) cell lines using a commercially available kit (Cell Titer 96 Aqueous One Solution Cell Proliferation assay, Promega). Cells were seeded in a 96 multiwell plate at a density of 2.5 × 104 cells/well and grown in Dulbecco’s Minimum Essential Medium (DMEM) with 10% FBS (foetal bovine serum) and 1% of penicillin/streptomycin (10000 U mL-1 penicillin and 10 mg mL-1 streptomycin) at 37 °C in 5% CO2 humidified atmosphere. After 24 h the medium was replaced with 200 µl of fresh culture medium containing either RGO/CJ nanosystem (1 : 1 w/w) or RGO/CJ-PEGBT nanosystem (1 : 1 w/w) or CJ or CJ-PEGBT at different concentrations, corresponding to equivalent concentrations of doxorubicin hydrochloride equal to 5, 10, 15, 20, 25, 30 and 50 µM. RGO alone at concentration per well ranging from 21 to 210 µg mL-1 , equivalent to RGO contained in the nanosystems, was seeded as negative control. After 4 h, 24 h and 48 h, samples were taken away from the wells, and substituted by fresh medium (100 µL) and 20 µL of a MTS solution. Cells were incubated for additional 2 h at 37°C, and then the absorbance at 490 nm was measured using a microplate reader (Multiskan, Thermo, UK). Doxorubicin hydrochloride solutions at the same concentrations were used as a positive


Page 9 of 30

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

Biomacromolecules

control, as pure cell medium was used as a negative control. Results were expressed as percentage reduction of the control cells. All culture experiments were performed in triplicates. In vitro evaluation of the hyperthermic effect. The feasibility of the hyperthermia treatment was established on MCF-7 cell line irradiating cells treated with the selected RGO/CJ-PEGBT nanosystem with a 810 nm surgical diode laser with the power fitted at 7x10-2 W mm-3. In particular, the hyperthermic effect, directly measured as a reduction of cell viability, was evaluated simulating the acute effect mediated by thermoablation and the chronic effect triggered by hyperthermia (i.e., drug release and drug sensitization). Thermoablation treatment (acute). For these experiments, cells were seeded in 96 well plate at a density of 2.5×104 cells per well and grown in supplemented DMEM, as above described. After 24 h cells were incubated for 4 h with fresh medium containing RGO/CJ-PEGBT nanosystem (1 : 1 w/w) at concentration of 0.37 mg mL-1, corresponding to 10 µM of doxorubicin hydrochloride. For the fluorescence microscopy cells were irradiated with a 810nm laser beam for 300 sec (spot width, 1.2 mm; power, 7x10-2 W mm3). The medium was then removed and the cell monolayer was washed twice with DPBS and fixed with 4% formaldehyde for 15 min at room temperature. Subsequently, the formaldehyde solution was removed, the cells washed with DPBS and incubated with 200 µl of 0.1% Triton X-100 in PBS for 5 min. Finally, the cytoskeleton of cells were stained with Alexa Fluor® 647 Phalloidin and the nuclei with 4ˈ,6-diamidino-2-phenylindole (DAPI). The images were recorded using an Axio Cam MRm (Zeiss). For the cytotoxicity assay cells were irradiated with a 810nm laser beam for different time, ranging from 5 to 300 seconds. After the laser treatment, DMEM was removed, cells washed up twice with DPBS and cells viability evaluated with MTS assay, as above described. Laser treated cells were used as a negative control. Hyperthermia-triggered effects (chronic). To evaluated the hyperthermia-triggered effects in chronic, apart from the fact that the MTS assay was performed after 24 h of post-incubation following the laser


Biomacromolecules

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

Page 10 of 30

treatment, an analogous procedure to that reported for the acute experiment was adopted. Cells treated with the same surgical diode laser, at the same power and for the same time (from 5 to 300 seconds), were used as negative control. All results were expressed as percentage reduction of the control cells. All culture experiments were performed in triplicates. RESULTS AND DISCUSSION Synthesis of the conjugates with or without biotin pendants (CJ and CJ-PEGBT). The preparation of an inulin-based pH-sensitive copolymer (CJ), carrying high dose of doxorubicin covalently linked, was firstly carried out following a protocol already described.16 Briefly, the hydroxyl groups of α-Dglucopyranosyl-[β-D-fructofuranosyl](n-1)-D-fructo furanoside (Inulin) were partially derivatized with ethylenediamine (EDA) to provide primary amine pendants available for additional functionalizations. These amine functions were exploited in order to introduce in turn C5-alkyne and citraconic acid moieties by coupling the carboxyl group of pentynoic acid (P) in the presence of a mixture of EDC and NHS as activating agents and citraconic anhydride, respectively. These two steps were performed in tandem without prior purifications, since the by-products produced in the first step of the reaction did not interfere in the nucleophilic reaction taking place between the amine groups of INU-EDA-Pentyne and citraconic anhydride. The derivations degrees in EDA, P and C calculated are reported in Table 1. For comparative purposes the INU-EDA-P,C intermediate was then used to synthetize two polymer-doxorubicin conjugates, with and without biotin side chain as targeting agent, proceeding along two synthetic pathway. In the first approach INU-EDA-P,C-Doxo (CJ) was obtained as reported.17 After purification and isolation, the relative derivatization degree of doxorubicin (DDdoxo, 8.38 %), namely the percentage of linked doxorubicin with respect to the repeating units of inulin, was attained combining 1H NMR spectroscopy and UV spectrophotometry (See Table 1).


Page 11 of 30

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

Biomacromolecules

Scheme 1. Schematic representation of the synthesis of INU-EDA-P-PEGBT,C-Doxo (CJ-PEGBT): i) PNPC, DMF, microwave 25 W, 1h, 50 °C; ii) EDA, 1h, r.t.; iii) 4-pentynoic acid, EDC, NHS, water, 18 h, r.t.; iv) citraconic anhydride, THF, 2 h, r.t.; v)CuSO4, ascorbic acid, N3-PEG-BT, EDC, NHS, doxorubicin hydrochloride, pH 6.8, H2O/DMF 57:43, 18h, N2, r.t. INU-EDA-P,C had one repeating unit per chain functionalized with alkyne group helpful to perform biotinylation via the well-known Husgen 1,3-dipolar cycloaddition (Scheme 1), and besides, it carries a large number of carboxyl functions which may orthogonally react with doxorubicin if conveniently activated with a EDC/NHS mixture.


Biomacromolecules

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

Page 12 of 30

Similarly to folate, biotin is a cell growth promoter which is contained in cancer cells at higher concentration, owing to the over-expression of its specific receptor on the cell membrane.19 Several studies showed indeed that biotin-conjugated carriers can significantly increase the uptake of anticancer drugs in tumor cells.20,21 Hence, with the aim of improving the selectivity of the CJ conjugate towards cancer cells and its therapeutic power as anticancer prodrugs, we have considered a biotin modification reagent (N3-PEG300-biotin) containing a discrete PEG spacer arm and a chemoselective reacting group at the other end which might react with the alkyne function as well in the presence of activated carboxyl groups. This PEG chain was selected because provides a molecular length after conjugation of ≈ 24 Å, so that a suitable modification of the original architecture can be accomplished while keeping a good solubility in aqueous media. The 1,3-dipolar cycloaddition involved a slight excess N3-PEG300-biotin to complete the alkyne depletion and copper (II) as catalyst. Even if this cycloaddition can be performed thermally, without adding the catalyst, the conditions usually used (T > 60°C) bring about change in regioselectivity and affect polymer and drug integrity thus leading to unsuitable polymeric architectures. Anyway, copper (II) was completely removed in bulk using a cation exchanging resin, which was filtered after that a colorless solution was obtained. After purification by exhaustive dialysis, the structure of CJ-PEGBT was confirmed by 1H NMR spectra where the appearance of a peak at δ 3.62 relative to the CH2O and CH2N hydrogens of the PEG arm, together with the peak at δ 2.50-2.60 ascribable to the SCH2 and NHCOCH2 hydrogens (BT), displays that biotinylation occurred.

The PEGBT mol% derivatization (DDPEGBT%) was found applying an

iterative method, being the PEG’s signal overlapped to that of the inulin hydrogens. However, the integral of the PEG hydrogens were calculated considering that they were exactly three times bigger than that of BT hydrogens at δ 2.50-2.60 and, thus the DDPEGBT% was calculated using this equation:

( .. /4) 100

[ .. − ( .. 3)]/7 Where Aδ 2.50-2.60 and Aδ 3.50-4.60 are the integrals of the peaks attributable to the BT and PEG plus inulin hydrogens respectively. 1H NMR spectra also hinted that the molar derivatization degree of doxorubicin


Page 13 of 30

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

Biomacromolecules

(DDdoxo) was roughly 5.9 %, not so far from the value obtained by UV spectrophotometry (6.02 %), pointing out that the one-pot reaction successfully befell. The weight average molecular weight (Mw) and the polydispersity (PD) of the conjugates were extrapolated by SEC traces obtained using an higher saline Tris buffer as eluent to promote solventpolymer interactions. Data are reported in Table 1. It might be noticed that the molecular weight of the copolymers coherently increases with the degree of functionalization, passing from 4.1 kDa for the pure inulin to 6.8 kDa for the CJ-PEGBT respectively. In addition, the Mw of CJ-PEGBT increases only by 600 Da (6.8 kDa vs 5.9 kDa), which corresponds more or less to a PEG-BT pendant, thus endorsing the structural hypothesis obtained from the 1H NMR spectra and UV data. Preparation and characterization of the nanosystems. Graphene-based nanostructures are state-of-theart technology to provide both excellent anticancer drug delivery and hyperthermia, combining biodegradability23,24 and versatility. The wide surface of the RGO used in this work (2.4 x 105 nm2 per macromolecule) enables the adsorption of large amount of molecules which can be properly released by established stimuli, thus leading to a local therapeutic effect with negligible side effects. Attempts were made to prepare stable RGO-based nanosystems bearing high amount drug-conjugates, both with and without biotin as targeting agent. To achieve this the idea was to facilitate π-staking interactions between the huge π-π conjugated structure of RGO and the doxorubicin moieties of the CJ and CJ-PEGBT conjugates. The characterization of the system without biotin (RGO/CJ-PEGBT) is reported in the supporting information, yet the essential features are discussed here to make a comparison with the biotinylated nanosystem. The virgin RGO had a pH of about 4.1, where its carboxylic acid were partially deprotonated. At this pH value doxorubicin hydrolysis from the conjugates may occur. So, when used to adsorb the conjugates onto its surface the pH of the RGO dispersion was adjusted near the neutrality (pH 7.2). A π-staked nanosystem was obtained after adding a conjugate solution with sonicating, stirring and incubating the resulting dispersion in the fridge overnight. Preliminary experiments were carried out using a


Biomacromolecules

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

Page 14 of 30

RGO/conjugate ratio (w/w) from 1:1 to 1:5. Only the nanosystems obtained with equivalent amount of both components were stable to the naked eye, without dissimilarity between the biotinylated and nonbiotinylated one. As CJ and CJ-PEGBT became more abundant in the solution, the risk of flocculation increased proportionally.

Table 1: Molar derivatization degree values of EDA, P, C, PEG-BT and Doxo linked to inulin; average weight molecular weight (Mw), polydispersity and ζ-Potential of the conjugates and their parent compounds. Sample

Mw

Mw/Mn

Composition

ζ-Pot

(kDa)

(mV) a

DDPentine

b

(mol %)

(mol %)

(mol %)

(mol %)

(mol %)

DDEDA

a

DDcitric.

b

DDDoxo

c

DDPEGBT

INU-EDA

4.1

1.87

18.0

-

-

-

-

18.1±3.1

INU-EDA-P,C

4.4

1.70

18.0

3.6

14.1

-

-

-25.3±2.5

CJ

6.2

1.61

18.0

3.6

14.1

8.38

-

-18.9±5.4

CJ-PEGBT

6.8

1.48

18.0

3.6

14.1

6.02

3.5

-21.7±5.1

a

Calculated by 1H NMR spectroscopy.\

b

Calculated combining 1H NMR spectroscopy and UV spectrophotometry.

c

Calculated combining 1H NMR spectroscopy and HABA dye assay.

Given this knowledge of the stability of these RGO/conjugate nanosystems, a fixed weight ratio was selected in the various experiments. The π-staking interactions between RGO and both conjugates were assessed by differential scanning calorimetry (Figure 1 and Figure S2). Figure 1 shows the thermograms obtained for RGO, CJ-PEGBT and the couple RGO/CJ-PEGBT. RGO alone displays an exothermic peak at 220 °C owing to decomposition of its carboxylic functions, as CJ-PEGBT had a similar peak of thermal decomposition at 260 °C. The thermogram obtained with the RGO/CJ-PEGBT nanosystem still kept the typical peak of RGO at 220 °C together with a small peak at 295 °C. The simultaneous disappearance of


Page 15 of 30

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

Biomacromolecules

the peak at 260 °C and the smaller enthalpy of the transition observed at 220 °C (602.6 J g-1 vs 1460 J g-1) arise due to strong interactions between the conjugate and RGO surface, which stabilize each other.

Figure 1 Chemical interaction study between RGO and CJ-PEGBT by differential scanning calorimetry (DSC): RGO (black line), CJ-PEGBT (red line) and nanosystem (fuchsia) A similar trend was observed for the couple RGO/CJ, but with some meaningful difference (Figure S2). In particular, the enthalpy of the transition observed at 220 °C was smaller (419.5 6 J g-1 vs 602.66 J g-1) suggesting the formation of a more collapsed nanostructure which further thermally stabilize the organic functions of the RGO perimeter. Another approach to study such physical interactions is to observe the rheological behavior of the nanosystem likening to the plain conjugate and RGO at the same concentration. From the analysis of the stress vs shear rate curves obtained applying both an increasing and then a decreasing stress (up and down ramp), two occurrences can be elicited, namely the formation of new structural arrangements (thixotropy) and the flow behavior. Figure 2 (A, B and C) shows that all samples behaved as a quasi-Newtonian fluid with a shear rate-dependent flow, but some difference deserve further considerations. In particular, while for CJ-PEGBT and the plain RGO a reversible thixotropic evolution arising from normal coil extensions and particle alignments was observed, for the RGO/CJ-PEGBT nanosystem a Newtonian-Bingham transition was revealed at high shear rate (Figure 2 C). According to the DSC data, this evokes the formation of π-staked systems arranged into aligned and hierarchical architectures owing to the polymer


Biomacromolecules

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

Page 16 of 30

chain-RGO platelet entanglement. In another set of experiments the viscosity of RGO, CJ-PEGBT and the RGO/CJ-PEGBT nanosystem were measured using a shear rate in which they behave as Newtonian fluids (10 rad sec-1) allowing to calculate time-independent values. As expected the viscosity of the nanosystem was roughly the same as measured for the graphene dispersion (ηRGO= 1.28 10-3 Pa s; ηRGO/CJ-PEGBT= 1.32 10-3 Pa s), implying that the rheometer experienced only the contribution of the graphene platelets on the viscosity instead of the polymer solution (1.45 10-3 Pa s). As polymer chains were adsorbed onto the RGO platelets they may not affect the viscosity of the PBS medium.

Figure 2. Stress vs shear rate curves obtained for CJ-PEGBT (A), virgin RGO (B) and RGO/CJ-PEGBT nanosystem (C) from 0.1 to 40 rad sec-1: up ramps (solid symbol) and down ramps (open symbol). The ability of the copolymer to lead to nano-sized system without any massive aggregation was also assessed by atomic force microscopy (AFM) analysis. AFM micrograph of the virgin RGO confirmed that it was well dispersed in single layer platelets of 1 nm thickness and 400±40 nm average width (Figure 3A). The complete adsorption of CJ-PEGBT onto RGO surface was highlighted by the increased thickness of the nanosystem, passing from 1 nm to 2 nm (Figure 3B). Notwithstanding the efficient adsorption of the conjugate onto RGO could cause aggregation, the resultant nanosystem appeared homogeneous and still dispersed implying a good stability in aqueous media (See also Supplementary data, Figure S4). Parallel results were obtained for the RGO/CJ based nanosystem.


Page 17 of 30

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

Biomacromolecules

Figure 3. AFM image of virgin RGO (A) and RGO/CJ-PEGBT nanosystem (B) Evaluation of the physicochemical properties of the nanosystems Study of the hyperthermic effect of RGO/CJ-PEGBT. The ability of RGO/CJ-PEGBT to convert a cold energy source into heat (hyperthermic effect) was investigated applying a 810 nm laser beam for 300 sec and measuring the dispersion temperature varia-

Figure 4. Hyperthermic effect of the RGO/CJ-PEGBT nanosystem in water: plotting of the temperature observed as a function of the exposure time at increasing concentrations of nanosystem (0.05 - 1 mg mL-1 from the bottom) (A); heating rate observed after later treatment vs concentration of the nanosystem (B)


Biomacromolecules

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

Page 18 of 30

-tion at scheduled time. The experiments were carried out at a concentration within the rage from 0.05 mg mL-1 to 1 mg mL-1 (Figure 4) so as to study the influence of this parameter on the hyperthermic effect. The dispersion temperature linearly increased administrating laser energy with fixed power and, as expected, the heating rate was strongly affected by the concentration of the nanosystem (Figure 4A). However the heating rate increased following a non-linear correlation affording a plateau at concentration of 1 mg mL-1 (Figure 4B). While until a concentration of 0.25 mg mL-1 it linearly increased, as the concentration of nanosystem further increased, the laser spot was completely saturated by RGO platelets and the amount of light absorbed did not significantly change reflecting in a slope reduction until reaching a plateau. In other words, one can deduce that, being the wavelength comparable to the RGO plates dimension (810 nm vs 500 nm), at concentration near 1 mg mL-1 each wave interacts with only one RGO plate maximizing the energy source transduction. Drug release study. The dialysis equilibrium time, previously determined employing equivalent amount of doxorubicin hydrochloride at both pH, was not dependent on the medium and was reached after 1.5 h. Figure 5A shows that biotinylated systems released doxorubicin more quickly than those nonbiotinylated, providing a good evidence that the presence of the PEGBT harm affects the arrangement of the polymer backbone leading to loose coils with greater solvent accessibility. For the RGO-based nanosystems a strong retention of doxorubicin can be noticed until to an incubation time of 12 h with a sharp leap of drug release after this time, displaying a remarkable time dependent release profile. As suggested by DSC and rheological data, this can be explained considering that the conjugates are stably adsorbed onto RGO, and so doxorubicin can be efficiently released only after a partial desorption of some polymer chains followed by hydrolysis of the citracolylamide bridges. In addition, for the RGO/CJPEGBT pair, where the lack of alkyne pendants imply less π- π interactions with the aromatic surface of RGO, a sustained doxorubicin release was observed with respect to the CJ-based nanosystem (8% vs 4 % respectively) without hinting at “burst effect.


Page 19 of 30

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

Biomacromolecules

After a topical administration inside a tumor mass, as the nanosystem diffuse throughout tumor and so inside cancer cells the pH typically decreases (from 7.4 to 5.5. inside lysosomes); therefore release experiments were also performed varying pH of incubation media so as to understand the fate of the nanosystem in vivo. In particular, samples were incubated at pH 7.4 for the first 12 h and then at pH 5.5 for the residual time. The release curves of the conjugates hint a pronounced dependence of drug release on pH (Figure 5A), reflecting the pH sensitivity of the citraconylamide spacer. As expected, after the first passage at pH 7.4, all samples steeply released their drug payload at acidic conditions, up to four times more. On the contrary, the decreased pH entailed a reduction of the release rate for the nanosystems. Being RGO highly carboxylated, a substantial protonation of its carboxylic groups at acidic condition provided flocculation, consistent with a reduced hydrolysis of doxorubicin pendants.

Figure 5.(A) Drug release curves obtained at 37 °C, pH 7.4 (dash lines, solid symbol) and changing the pH of the medium from 7.4 to 5.5 after 12 of incubation (solid lines, open symbol): CJ (star), CJ-PEGBT (triangle), RGO/CJ (circle), RGO/CJ-PEGBT (square). (B) Comparison between doxorubicin released after 4 h at pH 7.4 and after the same time at pH 7.4 with 300 sec of laser exposure. The hyperthermia-triggered drug release was studied irradiating a dispersion of RGO/CJ-PEGBT with a cold surgical laser beam for 300 sec and pleasing the resulting dispersion in a dialysis test tube for 4 h so as to evaluate doxorubicin release at equilibrium. It might be noticed that just after 4 h the amount of


Biomacromolecules

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

Page 20 of 30

doxorubicin released was five times bigger than that released from the untreated nanosystem, pointing out that hyperthermia can simultaneously stimulate drug release and thermoablation (Figure 5B). Biological characterization of the nanosystem In vitro evaluation of the cytotoxic effect. The in vitro cytotoxic effect of the two conjugates and the nanosystem derived from these, namely RGO/CJ and RGO/CJ-PEGBT, was evaluated on MCF-7 cells by MTS assay after an incubation of 4 h, 24 h and 48 h. Equivalent amount of virgin RGO and doxorubicin hydrochloride were used as negative and positive control respectively. Figure 6 shows the cell viability of MCF-7 cells treated with different amount of RGO and doxorubicin hydrochloride (5; 10; 15; 20; 25; 30; 50 µM) or equivalent amount of conjugates or nanosystems. RGO was clearly cytocompatible in the whole concentration range considered. A time-dependent and dose-dependent efficacy for all sample was observed, as cell viability decreased with the incubation time and increasing amount of doxorubicin. However, some crucial difference deserve further comments. In particular, the biotinylated samples, namely CJ-PEGBT and RGO/CJ-PEGBT, are always more effective than the parent samples. Just after 4 h of incubation the amount of cells killed by CJ-PEGBT was two times higher than that killed by CJ (Figure 6A). After 24 h of incubation, where cell viability at the maximum concentration was 40% and 80% for RGO/CJ-PEGBT and RGO/CJ respectively, this trend was more clear and appreciable even between the nanosystems remarking a critical role of biotinylation for expressing a good anticancer effect (Figure 6B). After an incubation time of 48 h, the biotinylated conjugate (CJ-PEGBT) had the same cytotoxicity profile than that observed for free doxorubicin hydrochloride, as CJ showed a similar evolution only at higher concentrations (> 25 µM) (Figure 6C).


Page 21 of 30

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

Biomacromolecules

Figure 6. Cytotoxicity assay on MCF-7 cell line after 4 h (a), 24 h (b) and 48 h (c) of incubation: RGO (dash dot line, solid square), Doxorubicin hydrochloride (dash dot line, open square), CJ ( solid line, open circle), RGO/CJ nanosystem (dot line, open circle), CJ-PEGBT (solid line, solid triangle), RGO/CJPEGBT nanosystem (dot line, solid triangle) On the other hand, while RGO/CJ-PEGBT displayed a cytotoxicity comparable to that of the free conjugate at concentration higher than 25 µM, the non-biotinylated nanosystem (RGO/CJ) provided a cell viability of 60% even at higher concentration (Figure 6C). Apart from the higher efficacy, the EC50 values calculated by the dose-response curves above discussed showed a notwithstanding higher potency for the biotinilated conjugate (EC5048hCJ = 15; EC5048hCJ-PEGBT = 50

RGO/CJ-PEGBT

23

22

Sample

incubation (10 µM – 99 % cell viability), and thus exposed to a 810 nm laser beam with a fixed power of 7x10-2 W mm-3 until to 300 sec. To evaluate if RGO/CJ-PEGBT can keep a good anticancer effect still


Biomacromolecules

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

Page 24 of 30

after the hyperthermia treatment, so as to avoid cell growth owing to the persistence of some cancer cells, we also evaluated cell viability after 24 h of post-incubation following the laser exposure. All data are reported in figure 8 with respect to the RGO hyperthermic effect used as positive control. It is quite clear that the hyperthermia-induced anticancer effect in acute (thermoablation) is higher for RGO/CJ-PEGBT, if compared with the virgin RGO, reaching a maximum effect at an exposure time of 200 sec. Though RGO allows to get to the maximum effect employing a lower exposure time (150 sec), this was significantly muffled (37% vs 60% cell viability for RGO/CJ-PEGBT and RGO respectively). This is more evident comparing the EC50 values, because RGO/CJ-PEGBT had a potency at least three times higher than that observed for the virgin graphene platelets (See Table 3). Not surprisingly, the experiment carried out with a post-incubation period after hyperthermia treatment, which establishes the long term effect of the nanosystem, evidenced that both RGO/CJ-PEGBT and RGO maintained its anticancer effect provoking an additional cell death. However, from a mere pharmacological standpoint the virgin RGO exhibited a lesser cytotoxic effect accompanied with lower potency (Table 3).

Figure 8. Cytotoxicity assay on MCF-7 cell line at equivalent concentration of doxorubicin hydrochloride of 10 µM: RGO (solid line, open symbol) and RGO/CJ-PEGBT nanosystem (solid line, solid symbol) after 4 h of incubation followed by laser treatments; RGO (dot line, open symbol) and RGO/CJ-PEGBT


Page 25 of 30

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

Biomacromolecules

nanosystem (dot line, solid symbol) after 4 h of incubation followed by laser treatments and 24 h of postincubation These experiments were also performed by considering only the dose of nanosystem which enters cells (See supplementary data, Figure S5 and S6).Whilst hyperthermia was slightly reduced for all samples and treatments, the behavior was similar confirming that the nanostructure can enter cells performing well as anticancer nanomedicine. These data describe a model in which the nanosystem provide manifold mechanisms, in acute and in chronic, giving rise synergic anticancer effect helpful to avoid recidivism. Indeed, in one hand the energy-transducing capability of the RGO component locally kills cancer cells by simply applying a cold laser beam, which can lead to thermoablation in combination with chemosensitization towards doxorubicin. On the other hand, the hyperthermia-triggered drug release allows to keep a suitable local drug concentration thus stunting cell evasion and proliferation in chronic.

Table 3: EC50 for RGO, RGO/CJ-PEGBT and free doxorubicin hydrochloride after 4 h of incubation with RGO/CJ-PEGBT at equivalent concentration of doxorubicin equal to 10 mM and laser treatment. EC50

EC50PC *

(sec)

(sec)

RGO

>300

130

RGO/CJ-PEGBT

125

105

Sample

*Obtained after an incubation time of 24 h following the hypertermia treatment

CONCLUSIONS We successfully established the functionalization of a known inulin-doxorubicin conjugate with a PEGbiotin harm, used as targeting agent, by a common Huisgen cycloaddition improving cell adhesion and preserving its effectiveness as anticancer prodrug. The structural identification was attained by 1H NMR


Biomacromolecules

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

Page 26 of 30

and UV analyses, which also revealed that a quantitative conversion of alkyne pendants to 1,2,3-triazole occurred. This biotinylated inulin-doxorubicin conjugate (CJ-PEGBT) was efficiently combined with RGO in order to obtain a nanosystem simultaneously endowed with multiple synergic anticancer effects and high specificity. Indeed, notwithstanding it was negatively charged, the conjugate was capable of generating stable supramolecules with RGO, exploiting the interactions between its aromatic side chains and those of RGO platelets. Our analysis was based on DSC, rheometry, AFM and drug release study, demonstrating that a π-staked graphene-based nanosystem with excellent drug payload can be obtained choosing a proper conjugate/RGO ratio. In this paper we illustrate that RGO/CJ-PEGBT, due to its outstanding hyperthermic effect and pH sensitivity, is able to physically burn cells and, at the same time, release doxorubicin if irradiated with a surgical cold laser leading to local and sustained cytotoxicity upon hyperthermia treatments. These results broadly support a mode of action in which the laser irradiation can supply a selective and direct surgical thermoablation of the tumor mass accompanied with hyperthermiatriggered drug release and sensitization.

ASSOCIATED CONTENT Supporting information available 1

H NMR spectrum of CJ-PEGBT, DSC of the CJ/RGO nanosystem, AFM image of CJ/RGO,

physicochemical stability of RGO/CJ-PEGBT dispersion in different media, uptake study of RGO/CJPEGBT,. hyperthermic effect of the RGO/CJ-PEGBT nanosystem after cell entering. This information is available free of charge via the internet at http://Pubs.acs.org/.

AUTHOR INFORMATION Corresponding author *E-mail: [email protected] Fax: +39 09123891928;


Page 27 of 30

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

Biomacromolecules

Tel: +39 09123891928

AKNOLEDGEMENTS

We really must thank Dr. Francesco Carfì, Department of “Ingegneria Civile, Chimica, Ambientale e dei Materiali” (DICAM), for performing the DSC experiments reported in this paper.

REFERENCES

1 Juliano, R. Nat. Rev. Drug Discov. 2013, 12, 171-172. 2 Mura, S.; Nicolas, J.; Couvreur, P. Nat. Mater. 2013, 12, 991-1003. 3 Gottesman, N. M. Annu. Rev. Med. 2002, 53, 615-27. 4 Fuller, K. J.; Issels, R. D.; Slosman, D. O.; Guillet, J. G.; Soussi, T.; Polla, B. S. Eur J Cancer 1994, 30A(12), 1884–1891. 5 Chatterjee, D. K.; Diagaradjane, P. and Krishnan, S. Ther. Deliv. 2011, 2(8), 1001-1014. 6 Bass, H.; Moore, J.L.; Coakley, W.T. Int J Radiat Biol Relat Stud Phys Chem Med. 1997, ;33(1), 57–67. 7 Urano, M.; Kuroda, M.; Nishimura, Y.; Int J Hyperthermia 1999, 15(2), 79–107. 8 Poon, Ronnie T. P and Borys, N. Future Oncol. 7 (8), 937-945, doi: 10.2217/fon.11.73. 9 Vila, M.; Matesanz, M. C.; Gonçalves, G.; Feito, M. J.; Linares, J.; Marques, P. A. A. P.; Portolés M. T. and Vallet-Reg, M. Nanotechnology 2014, 25 (3), 035101. 10 L. Jingquan, L.; Liang, C.; Dusan, L. Acta Biomater. 2013, 9(12), 9243-9257. 11 Liu, Z.; Robinson, J. T.; Sun, X. M.; Dai, H. J.; J. Am. Chem. Soc. 2008, 130, 10876–10877. 12 Sun, X. M.; Liu, Z.; Welsher, K.; Robinson, J. T.; Goodwin, A.; Zaric, S.; Dali, H. Nano Res. 2008, 1(3), 203–212. 13 Zhou, T.; Zhou, X.; Xing, D.; Biomaterials, 2014, 35, 4185-4194.


Biomacromolecules

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

Page 28 of 30

14 van der Zee, M.; Stoutjesdijk, J. H.; van der Heijden, P. A. A. W.; de Wit, D. J. Environ. Polym. Degrad. 1995, 3, 235-242. 15 Licciardi, M.; Scialabba, C.; Sardo, C.; Cavallaro, G.; Giammona, G. J. Mater. Chem. 2014, 2, 4262–4271. 16 Scialabba, C.; Licciardi, M.; Mauro, N.; Rocco, F.; Ceruti, M.; Giammona, G. Eur. J. Pharm. Biopharm. 2014, 88, 695–705. 17 Mauro, N.; Campora, S.; Scialabba, C.; Adamo, G.; Licciardi, M.; Ghersi, G.; Giammona, G. RSC Advances 2015, 5, 32421-32430. 18 Vervoort, L.; Vinckier, I.; Moldenaers, P.; Van den Mooter, G.; Augustijns, P.; Kinget, R. J. Pharm. Sci. 1999, 88 (2), 209-214. 19 Russell-Jones, G.; McTavish, K.; McEwan, J. J. Inorg. Biochem. 2004, 98, 1625e1633. 20 Cannizzaro, S. M. Biotechnol. Bioeng. 1998, 58, 529-535. 21 Yang, W.; Cheng, Y.; Xu, T.; Wang, X.; Wen, L. Eur. J. Med. Chem. 2009, 44, 862-868. 22 Tripodo, G.; Mandracchia, D.; Collina, S.; Rui, M.; Rossi, R. Med Chem. 2014, S1-004, doi: 10.4172/2161-0444.S1-004 Open access. 23 Kotchey, G. P.; Allen, B. L.; Vedala, H.; Yanamala, N.; Kapralov, A. A.; Tyurina, Y. Y.; KleinSeetharaman, J.; Kagan, V. E.; Star, A. ACS Nano 2011 5(3), 2098‐2108. 24 Russier, J.; Ménard-Moyon, C.; Venturelli, E.; Gravel, E.; Marcolongo, G.; Meneghetti, M.; Doris, E.; Bianco, A. Nanoscale 2011, 3(3), 893‐896. 25 Droumaguet, B. L.; Nicolas, J.; Brambilla, D.; Mura, S.; Maksimenko, A.; De Kimpe, L.; Salvati, E.; Zona, C.; Airoldi, C.; Canovi, M.; Gobbi, M.; Noiray, M.; La Ferla, B.; Nicotra, F.; Scheper, W.; Flores, O.; Masserini, M.; Andrieux, K. and Couvreur, P. ACS Nano 2012, 6, 5866-5879. 26 Huang, Y.; Yang, Y.; Yang, K.; Shieh, H.; Wang, T.; Hwu, Y. and Chen, Y. BioMed Research International 2014, doi.org/10.1155/2014/182353.


Page 29 of 30

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

Biomacromolecules

27 Chen, A.C.; Huang, Y.Y:; Sharma, S.K.; Hamblin, M.R. Photomed Laser Surg 2011, 29(6), 383389. 28 Huang, Y.Y:; Nagata, K.; Tedford, C.E.; Hamblin, M.R. J Biophotonics 2014, 7(8). 656-664.


Biomacromolecules

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

Page 30 of 30

For table of contents use only

Biotin-containing Reduced Graphene Oxide-based Nanosystem as a Multi-effect Anticancer Agent: Combining Hyperthermia with Targeted Chemotherapy Nicolò Mauroa, Cinzia Scialabbaa, Gennara Cavallaroa, Mariano Licciardiab, Gaetano Giammonaab*

Graphic Table of Content Here the RGO-induced hyperthermia was combined with the stimuli-sensitive anticancer effect of a biotinylated inulin-doxorubicin conjugate (CJ-PEGBT), getting to a nanosystem endowed with synergic anticancer effects and high specificity.


Biotin-Containing Reduced Graphene Oxide-Based Nanosystem as a

Recommend Documents