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Targeted Delivery of Docetaxel Using Transferrin/Poly (allylamine hydrochloride)-Functionalized Graphene Oxide Nanocarrier Fatemeh Nasrollahi, Jaleh Varshosaz, Abbas Ali Khodadadi, Sierin Lim, and Ali Jahanian-Najafabadi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b02790 • Publication Date (Web): 09 May 2016 Downloaded from http://pubs.acs.org on May 14, 2016

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ACS Applied Materials & Interfaces

Targeted Delivery of Docetaxel Using Transferrin/Poly (allylamine hydrochloride)Functionalized Graphene Oxide Nanocarrier Fatemeh Nasrollahi a,b,c, Jaleh Varshosaz b,*, Abbas Ali Khodadadi a,*, Sierin Lim c, Ali Jahanian-Najafabadi d a

Catalysis and Nanostructured Materials Laboratory, School of Chemical Engineering, College

of Engineering, University of Tehran, Tehran, Iran, P.O. Box: 11155/4563 b

Novel Drug Delivery Systems Research Centre, Department of Pharmaceutics, School of

Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences and Health Services, Isfahan, Iran, 81746-73461 c

Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang

Technological University, 70 Nanyang Drive, Block N1.3, Singapore 637457 d

Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical

Sciences,, Isfahan University of Medical Sciences and Health Services, Isfahan, Iran, 8174673461 KEYWORDS: Graphene oxide, Docetaxel, Drug delivery, Transferrin, Poly (allylamine hydrochloride), Cytotoxicity, MCF-7

ACS Paragon Plus Environment

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ABSTRACT: The exceptional chemical and physical properties of graphene oxide (GO) make it an attractive nanomaterial for biomedical applications, particularly in drug delivery. In this work we synthesized a novel, GO-based nanocarrier for the delivery of docetaxel (DTX), a potent hydrophobic chemotherapy drug. The GO was functionalized with transferrin (Tf)-poly (allylamine hydrochloride) (PAH), which provided targeted and specific accumulation to extracellular Tf receptors, and stabilized GO in physiological solutions. Tf was conjugated to PAH via amide covalent linkages and Tf-PAH coated the surface of DTX-loaded GO through electrostatic interactions. The morphology and structure of the resulting nanostructure along with its surface modifications were verified using FT-IR, UV-vis spectroscopy, AFM, and SEM. DTX was loaded at a relatively high loading capacity of 37% and released at a pH-dependent and sustained manner at the physiological conditions. The targeting efficiency and cytotoxicity of this drug delivery system were evaluated on MCF-7 breast cancer cells. Improved efficacy of targeted DTX-loaded nanocarrier was observed compared to non-targeted one and free DTX especially at high drug concentrations. The Tf-PAH-functionalized GO nanocarrier is a promising candidate for targeted delivery and controlled release of DTX.

INTRODUCTION Taxanes family of drugs, including docetaxel (DTX, Taxotere) and paclitaxel (PTX, Taxol) has emerged as the most promising chemotherapeutic agents against a wide range of tumors. Taxanes have shown substantial clinical efficacy for the treatment of ovarian, colon, head and neck, non-small cell lung, and advanced breast cancers.1-3 Their cytotoxic mechanism is through inhibiting microtubule depolymerization and consequently inhibiting cell proliferation.4 DTX is


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a semi-synthetic analogue of PTX, extracted from needles of the European yew tree (Taxus baccata L.).5 It is significantly more efficacious than PTX against human ovarian, endometrial, colon, and breast cancer cell lines.6 Compared to PTX, DTX is more effective in inhibiting microtubule depolymerization with better anti-cancer activity, pharmacokinetic profile, and water solubility.7,8 Even though it has improved water solubility compared to PTX, DTX is practically insoluble in water and the only FDA-approved DTX products are formulated with high concentration of Tween 80 diluted in ethanol. This formulation is associated with severe side effects, including hypersensitivity reactions, cumulative fluid retention, nausea, mouth sores, hair loss, peripheral neuropathy, and anemia as well as incompatibility with commonly used polyvinyl chloride intravenous administration sets.9-11 Thus, despite the efficacy of DTX, the side effects arising from its poor water solubility has limited its clinical application and more effective methods are required for better delivery of DTX. In some investigations alternative formulations of DTX without or with low concentration of Tween 80 have been proposed. Nanoparticles-based drug delivery vehicles such as liposomes, solid lipid nanoparticles, polymeric nanoparticles, micro emulsions, micelles, and nanotubes have been introduced to carry and to deliver DTX.12-15 The stabilization of these nanoparticles in aqueous solutions will remove the Tween 80 requirement. Enhanced permeation and retention (EPR) effect of nanoparticles increases the concentration of drug in tumors and they show good tumor vasculature extravasation. Moreover, nanoparticles can be designed to have a longer circulation time in blood, targeting effect and controlled drug release.16


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The loading of different aromatic drugs on nanocarbons such as carbon nanotubes and graphene with sp2 domains is achieved through π–π stacking during a simple mixing process.17-20 Graphene oxide (GO) has been one of the most proposed nanocarriers for drug delivery in the past decade. Its one-atom thickness and two-dimensional plane consists of sp2-hybridized carbon atoms.21-22 The oxygen containing functional groups on the surface of GO facilitates its surface functionalization using targeting agents or polymers. As a result of its remarkable physical and chemical properties, graphene has attracted tremendous attention in biological and nonbiological applications.21,23-25 Potential applications of graphene and its derivatives in cellular imaging, photo thermal therapy, gene delivery, drug delivery and MRI have been extensively developed.17,18,26-28 In 2008, Dai et al. first reported the use of PEGylated GO (GO-PEG) for the delivery of SN38 as a hydrophobic anti-cancer drug and demonstrated the unique ability of GO-PEG in loading and delivery of aromatic water insoluble drugs.17 They found that in contrast to GO, GO-PEG is stable in cell medium and it is cytocompatible. In the same year, Yang et al. investigated the loading and release behavior of doxorubicin (DOX) on the surface of GO.29 They achieved drug loading capacity of 200%, which is much higher than that of other nanocarriers with loading capacity of less than 100%. They suggested GO as an efficient nanocarrier with high drug loading capacity To enhance the stability of GO nanoparticles in physiological environments, they were functionalized with hydrophilic and biocompatible polymers, which could also reduce GO toxicity.17,18,30-37 The covalent binding of PEG to the surface of GO has been widely used to improve biocompatibility and stability of GO.17,18,30,36,37 Xu et al. reported successful application of GO-PEG for delivery of PTX as a water insoluble drug.30,31 Some investigations introduced


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chitosan/GO nanocomposites for biological applications.32,35 Depan et al. enhanced the stability of GO in aqueous solutions using water soluble chitosan and prepared folic acid decorated nanostructure for the delivery of DOX to folic acid receptors.35 Considering the importance of targeted nanoparticles in reducing side effects of drugs, improving drug efficiency, and decreasing the required dose, conjugation of GO with targeting agents has been remarkably studied. Dai et al. prepared photoluminescent ultrasmall nanographene oxide (nGO) sheets (~10 nm), targeted with Rituxan antibody for selective binding to B-cell lymphoma cells and cellular imaging.18 Targeting of GO particles using folic acid was introduced by Zhang et al. for targeted delivery of mixed anti-cancer drugs,19 and it was later developed in other investigations.28,35 Liu et al. investigated the delivery of DOX using transferrin targeted GO-PEG and higher cytotoxicity and delivery efficiency of targeted GOPEG to C6 glioma cells were evidenced.37 Integration of GO and magnetic nanoparticles was also studied for site directed drug targeting.38 Herein, we present a GO-based nanocarrier to be used for delivery of DTX in an aqueous formulation. This nanocarrier can be used for delivery of high concentration of DTX without the side effects arising from its poor water solubility. To prepare a stable nanocarrier in physiological environments, drug loaded GO (GO-DTX) was non-covalently functionalized with poly (allylamine hydrochloride) (PAH) as it is cationic, highly water soluble, and biocompatible polymer.39 PAH has been extensively used in biological applications and it is most frequently employed in preparation of multilayers or nanocapsules using layer-by-layer method for gene and drug delivery purposes.40,41 Our proposed nanocarrier is also decorated with transferrin (Tf) to enhance targeting efficiency and DTX delivery to MCF-7 human breast cancer cell line. Tf is


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a biodegradable, nontoxic, non-immunogenic, and effective tumor-targeting agent due to the high expression of Tf receptors on tumor cell surfaces.42,43 As shown in scheme 1, GO dispersion in water was prepared by oxidation of graphite followed by ultrasonication. The GO surface was physically loaded with DTX with the aid of aromatic rings and functional groups in the structure of GO and DTX. After the conjugation reaction and formation of amide covalent linkage between Tf and PAH, the GO-DTX was functionalized with synthesized Tf-PAH through electrostatic interaction. It was found that the targeted DTX-loaded nanocarrier could specifically deliver DTX to MCF-7 breast cancer cell line via Tf receptors and showed significantly higher cytotoxicity compared to non-targeted DTX-loaded nanocarrier.


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Scheme 1. Steps for preparation of Tf-PAH-(GO-DTX) complex


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EXPERIMENTAL SECTION Materials. Graphite powder, dimethyl sulfoxide (DMSO), H2SO4, H3PO4, H2O2, HCl and HPLC grade acetonitrile and methanol were purchased from Merck Chemical Co (Germany). Docetaxel (DTX), human Tf, poly (allylamine hydrochloride) (PAH~15 kDa), 1-Ethyl-3-[3(dimethylamino)propyl]carbodiimide hydrochloride (EDC) and N-Hydroxysuccinimide (NHS) were purchased from Sigma (USA). The dialysis membrane (molecular mass cutoff of 20 kDa, 10 kDa) was obtained from SpectrumLabs Co (USA). Amicon ultra centrifugal filter units (100 kDa and 10 kDa) were from Merck Millipore (Ireland). Fetal bovine serum (FBS), RPMI 1640 culture medium, 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA), penicillin streptomycin (Pen Strep) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Cinnagen (Iran). The MCF-7 breast cancer cell line was a gift from professor Zarkesh, Institute of Biology of Isfahan University (Iran). Ultrapure water was used in all experiments. Instrumentation. The morphology of GO-based samples was measured using transmission electron microscopy (TEM, Philips EM 208, USA) and scanning electron microscopy (SEM, JEOL JSM 6701F, Japan). The atomic force microscopy (AFM) in tapping mode (Park Systems XE100, Korea) was employed to take images from samples dropped on mica substrate. The size distribution and ζ potential of nanoparticles were measured using a Nanosizer (Zetasizer, Malvern, UK) based on the dynamic light scattering (DLS) technique. The HPLC analysis was performed using an HPLC (Waters 2487, USA) instrument, to measure concentration of DTX in the solutions. The UV absorption and Raman spectra were recorded with a UV-vis spectrophotometer (Shimadzu UV 2450, Japan) and a Raman spectrophotometer (Senterra Bruker, USA). The FT-IR (Bruker Vector22, USA) was employed to collect the FT-IR spectra of


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samples in KBR pellets. The GO sheets were exfoliated and sonicated using a probe ultrasonicator (Heilscher UIS250 V, USA). Preparation of GO. GO was prepared according to an improved Hummers’s method called Tour's method44 which produces more oxidized and hydrophilic GO, compared to Hummers’s method.45 While stirring, KMnO4 (6.0 g) was gradually added to a mixture of graphite powder (1.0 g), concentrated H2SO4 (90 ml) and H3PO4 (10 ml). The reaction mixture was heated to 50°C, and maintained at this temperature for 12 h. The deionized water (100 ml) was then added to the reaction mixture, followed by addition of 30% H2O2 (3 ml) to produce a yellow color product containing oxidized graphite. The mixture was centrifuged and the precipitated solid was washed several times with deionized water (150 ml), 30% HCl (150 ml) and ethanol (150 ml) until the pH of the supernatant became neutral and finally it was dispersed in deionized water. After ultrasonication for 2 h, using a probe sonicator, the product was centrifuged at 10000 rpm (11000 ×g) for 30 min and the supernatant which contained exfoliated small GO sheets was collected. This homogenous dispersion of GO sheets in deionized water showed high stability and has not precipitated for several months. Synthesis of Tf-PAH Bioconjugate. An EDC mediated reaction was carried out to conjugate Tf with PAH through formation of amide linkages between COOH groups of Tf and NH2 groups of PAH.46 Briefly, a solution of Tf (10 mg) in 10 mM phosphate buffer solution at pH 7.4 was prepared, followed by addition of EDC (24 mg) and NHS (14 mg) as the coupling reagents to activate the carboxyl groups of Tf. The resulting solution was gently stirred for 2 h under nitrogen atmosphere at 10ºC in darkness. Subsequently, 50 mg of PAH was added to the solution and the reaction mixture was incubated under gentle stirring for 18 h in nitrogen atmosphere at 10ºC in the dark. The unreacted polymers, Tf, small molecules and ions were removed by


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dialysis in a dialysis bag (20 kDa) against deionized water for 24 h, followed by centrifugation using Amicon ultra-centrifugal filter devices (100 kDa). The filtrate was analyzed to determine the content of conjugated protein using Bradford protein assay. Finally, the concentrated retentate containing Tf-PAH conjugates was collected and freeze dried for further experiments and characterization tests. Loading of DTX on GO Sheets. A solution of GO dispersed in deionized water (4.5 ml of a 1 mg/ml) was added to DTX solution in DMSO (8 ml of a 1 mg/ml) to reach the DTX concentration of 0.66 mg/ml and GO concentration of 0.33 mg/ml. The mixture was stirred for 24 h at room temperature in darkness. The undissolved DTX was separated by centrifugation at 3500 rpm (1400 ×g) at which GO nanoparticles did not settle down. To remove the free DTX which was dissolved in the mixture, the supernatant was filtered through a 10 kDa molecular weight cutoff filter and the solution retained in the filter was washed repeatedly with water. Finally, the GO-DTX mixture and the filtrate containing free DTX were separately freeze dried. The concentration of loaded DTX was measured indirectly by analyzing the amount of unbounded DTX. The freeze dried sample containing free unbounded freeze dried DTX was dissolved in a suitable volume of acetonitrile to be injected to the HPLC column. Standard DTX calibration curve was then used to quantify unknown concentration of DTX. The amount of loaded DTX was calculated by subtracting this value from the initial amount of added DTX. The preliminary direct measurement of DTX amount loaded on the GO sheets showed no significant difference with the indirect method. Therefore, the indirect determination was used, due to its ease and feasibility. The experiment was repeated for DTX concentrations of 0.15, 0.3, 0.5 and 1 mg/ml at the same GO concentration (0.33 mg/ml), to investigate the effect of drug concentration on loading capacity of GO particles.


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HPLC Assay for DTX. The quantitative determination of DTX was performed using a high pressure liquid chromatography-UV system equipped with a reverse phase C18 column (4.6×250 mm, pore size 5 µm, Waters, USA). The mobile phase was acetonitrile-water (65:35) at flow rate of 1.0 ml/min and the detection wavelength was set at 230 nm. The injection volume for dug analysis was 40 µl and the run time was 10 min for each sample. The calibration curve was linear by analyzing a series of concentrations of DTX in acetonitrile in the range of 0.25–40 µg/ml with a correlation coefficient of R2=0.9995. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 30, 100 ng/ml respectively. Standard samples were prepared and injected in triplicate on three successive days. Preparation of Targeted DTX-Loaded Nanocarrier (Tf-PAH-(GO-DTX)). Solution of TfPAH conjugate (2 mg/ml) in 5 ml deionized water was added gradually under rapid stirring at room temperature in darkness to 5 ml of a 1 mg/ml of a GO-DTX suspension in deionized water. After stirring for 10 h, the product was ultracentrifuged at 25000 rpm (70000 ×g) for 1 h and washed several times with water, followed by lyophilization. The steps for preparation of TfPAH-(GO-DTX) are illustrated in scheme 1. In Vitro Drug Release. Freeze dried Tf-PAH-(GO-DTX) was dispersed in deionized water and divided into three equal aliquots. Each solution was placed in a dialysis bag and immersed in PBS (50 mM) at pH 7.4. The release reservoir was kept under permanent stirring, at the physiological temperature of 37°C. At different time intervals 0.5 ml of each release reservoir was withdrawn for HPLC analysis, followed by replacing the same amount of fresh PBS in each release reservoir. Each sample was centrifuged to remove undissolved solid particles and injected to HPLC column for analyzing the amount of drug released. The drug release experiment was repeated for pH 5.5.


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Cell Culture. The MCF-7 human breast cancer cell line was provided to investigate the cytotoxicity of DTX-loaded and blank nanocarriers. The cells were cultured in the flasks containing RPMI 1640 medium supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin, and incubated at 37ºC under a humidified 5% CO2 atmosphere. In Vitro Cellular Toxicity Assay. MTT assay was performed to investigate the cell viability of DTX loaded targeted (Tf-PAH-(GO-DTX)) and non-targeted (PAH-(GO-DTX)) nanocarriers, and blank nanocarrier (Tf-PAH-(GO)). The cells were seeded in the 96-well plates at a density of 7.5x103 cells per well in 200 µl culture medium and incubated for 24 h. The cell medium was then replaced with 180 µl of fresh medium and the cells were treated with 20 µl of Tf-PAH(GO), free DTX, PAH-(GO-DTX), Tf-PAH-(GO-DTX), and Tf-PAH-(GO-DTX)+Tf samples at final DTX concentrations of 0.03, 0.8, 3.5, and 8 µg/ml. The cell medium in free DTX samples contained 1% (v/v) DMSO. To study whether the drug uptake is carried out through the Tf receptors, the cell’s Tf receptors were saturated using 20 µl of Tf solution (5 mg/ml) 1 h prior to incubation with Tf-PAH-(GO-DTX) at DTX concentrations of 3.5 and 8 µg/ml. After continuous incubation for 24, 48, 72 and 96 h, 20 µl of 5 mg/ml MTT stock solution was added to the wells. The cells were incubated for another 3 h, followed by removal of cell medium and addition of 150 µl of DMSO to dissolve the formed formazan crystals. The absorbance of each well was recorded at 570 nm using a Bio-Tek microplate reader (PowerWave XS, USA) to determine the relative cell viabilities. Statistical Analysis. The data are presented as the mean ± standard deviations. The one-way analysis of variance (ANOVA) test was employed to analyze the statistical differences using the SPSS software (ver.18, USA). The P values of less than 0.05 were considered as statistically significant difference.


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RESULTS AND DISCUSSION Characterization of GO. GO was prepared by chemical oxidation of graphite followed by probe-ultrasonication to reduce the size of particles and to exfoliate GO sheets in the aqueous solution. The prepared GO dispersion was stable in water for more than six months and no precipitation was observed. This stabilization is a result of the existence of many oxygen containing groups including hydroxyl, epoxy and carboxyl on GO sheets. AFM was employed to characterize the thickness and morphology of GO nanoparticles. The sheet like structure of GO particles with a smooth surface is shown in Figure 1a. The height profile of GO sheets in the image confirms a thickness of about 1.0-1.6 nm corresponding to one to two layers of GO.24,47 The size distribution of GO obtained from AFM analysis in Figure 1b reveals lateral sizes of 40-100 nm for most GO sheets with an average of 76 nm. The TEM and SEM images also illustrate the sheet-like structure of GO particles. The disordered stacking of smooth graphene sheets is shown in SEM image in Figure 1c. The transparent ultrathin nanosheets in TEM image in Figure 1d illustrate the efficient exfoliation of nanosheets. Some bends and wrinkles can be observed on the surface of nanosheets after oxidation process. Raman spectroscopy was carried out at excitation wavelength of 785 nm to evaluate deformations in the structure of GO. The two most prominent peaks which are commonly observed in GO samples can be seen in Figure 1e. The G band at 1583 nm corresponds to the E2g vibrational mode of sp2 domains. Moreover, the relatively intense D band at 1329 nm indicates defected and disordered graphitic plane, which is a result of the oxidation process and the formation of oxidized functional groups on the surface and edges of GO sheets.48 The intensity ratio of D band to that of G band of 1.23 also indicates an increase of edge planes and disorders


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as well as decrease of the size of the sp2 domains of graphene sheets after the oxidation process.49,50

Figure 1. (a) AFM image of GO and the corresponding AFM height profile, (b) AFM size distribution of GO, c) SEM image of GO, (d) TEM image of GO, (e) Raman spectrum of GO.


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DTX Loading. DTX is a widely used aromatic anti-cancer drug that is insoluble in water. Leveraging on the simple physical loading of this aromatic drug on the surface of GO, and the stability of GO in aqueous solutions, we hypothesize that GO is a suitable nanocarrier for DTX. DTX was loaded on GO surface after mixing a solution of DTX in DMSO with GO solution in water, followed by separation of the free DTX by centrifugation and filtration. The prepared GODTX nanoparticles were readily dispersed in water. In contrast to many anti-cancer drugs which can be quantified using their UV-vis characteristic peak, the concentration of DTX was assayed by HPLC analysis because of its considerably low UV absorbance especially at low drug concentrations. The standard calibration curve of DTX obtained by HPLC analysis is shown in Figure 2a, which illustrates elution of DTX after 5 min. Considering the significant difference in the range of measured DTX concentrations in the release and loading experiments, two different standard curves at two ranges of DTX concentrations were obtained from HPLC analysis for each of the loading and release experiments. The loading of DTX on GO at different initial DTX concentrations with respect to the same concentration of GO (1 mg/ml) was evaluated and the results are shown in Figure 2b. The percentage of the weight ratio of DTX to GO in the GO-DTX sample, as the loading capacity of GO, increases with increasing the initial concentration of DTX, and reaches 37% at DTX concentration of 1 mg/ml. This high loading capacity can be explained by high aspect ratio and loading of DTX on both planes of GO. The efficient DTX loading can also be attributed to the strong π–π stacking between the aromatic rings of DTX and GO as well as hydrogen-bonding interactions between their functional groups. As shown in Table 1, the obtained loading capacity of GO is much higher than the reported DTX- loading capacity of less than 10% for commonly


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used nanocarriers like liposomes, micelles, polymeric, chitosan and solid lipid nanoparticle.51-55 Among the carbon-based nanocarriers, activated carbon nanoparticles exhibited low DTXloading capacity.55 However, the DTX-loading capacity of single-walled and multi-walled carbon nanotubes is usually higher than that of other nanocarriers and is comparable with GO. 5659

Similar to GO, this high loading capacity is attributed to the large surface area of CNTs and

can be optimized by increasing the initial concentration of DTX.58,59

Figure 2. (a) HPLC analysis of DTX at different DTX concentrations and calibration curves in two concentration ranges, (b) loading capacity of DTX on GO at different initial DTX concentrations and at GO concentration of 1mg/ml


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Table 1. Reported loading capacity (%) of DTX-loaded nanocarriers and percentage of released drug from their structures DTX- Loaded Nanocarrier

Loading Capacity (%)

Drug Release (%)

DTX-loaded chitosan51

8.9-12 %

68-83% (pH 7.4, 24h)

DTX-loaded pluronic P123 micelles52

2.12%

90% (pH 7.4, 50h)

DMAB-modified DTX- loaded PLGA nanoparticles53

9%

15% (pH 7.4, 120h)

Trimyristin-based, PEGylated DTX incorporated SLNsa,54

2.4-2.8 %

80-90% (pH 7.4, 150h)

DTX-loaded liposome55

4.25%

-

DTX-loaded modified activated carbon55

4.46%

-

DTX-loaded ultrashort oxidized SWNTsb (π–π stacking), conjugated to FA, mediated SLNs56

9.67%

40% (pH 7.4, 48h)

DTX-loaded SWNT (π–π stacking), conjugated to NGR Peptide and noncovalently functionalized with surfactants (PVPk30, Poloxamer 188, Phospholipids, HS 15)57

-Poloxamer: 5-15% -HS 15: 10-25% -Phospholipids: 10-35% -PVPk30: 20-120%

DTX-loaded oxSWNHsc (π–π stacking), noncovalently wrapped to PEG-antiVEGF antibody58

42-74% (Feeding DTX/oxSWNHS ratio of 5-16)

59% (pH 7.4, 144h)

DTX-loaded MWNTsd (covalent attachment)59

38-56% (Feeding DTX/MWNTs ratio of 0.125-1)

35% (pH 5, 16h) 27.5% (pH 6.8, 16h) 25 % (pH 7.4, 16h)

DTX-loaded MWNTs (π–π stacking)60

-

32.77% (pH 7, 8h) 42.67% (pH 4, 8h) 58% (pH 7, 72h) 68% (pH 4, 72h)

a

Solid Lipid Nanoparticles

c

Oxidized Single-Walled Carbon Nanohorns

b

Single-Walled Carbon Nanotubes

d

Multi-Walled Carbon Nanotubes


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To investigate the loading of DTX on GO, we performed UV-vis and FT-IR spectroscopies on DTX, GO and GO-DTX. Figure 3a shows a single broad UV absorption peak at 227 nm for GO, originating from the ߨ → ߨ ∗ transitions of the aromatic C=C bond.61 This characteristic peak was used for quantification of GO. Similar to GO, the GO-DTX displays a relatively high UV-vis absorbance in a wide range of wavelengths and the shift of characteristic peak of DTX from 223 nm in DTX to 208 nm in GO-DTX suggests loading of DTX on the GO surface. The FT-IR spectra of GO in Figure 3b, indicates the presence of oxygen-containing groups on the surface of GO. The broad peak at 3407 cm-1 is assigned to the O-H stretching vibrations, and the peaks at 1623 cm-1 and 1737 cm-1 correspond to the C=C vibrations of aromatic rings and the C=O vibrations of carboxyl groups, respectively. The peaks arising from the C-O stretching vibrations (carboxyl, epoxy and alcoxy groups) can also be observed at 1384 cm-1, 1170 cm-1 and 1097 cm-1.62 The FT-IR spectra of GO-DTX in comparison with GO and DTX were also examined. As shown in Figure 2b, the peaks of GO at 1623 cm-1 and 1097 cm-1, shift to higher positions at 1635 cm-1 and 1116 cm-1 in GO-DTX, which can be explained by the formation of hydrogen bonds and ߨ − ߨ stacking between DTX and GO. Moreover, the appearance of new peaks at 709 cm-1, and 1261 cm-1, which respectively belong to the out plane N-H vibration, and the C-N stretching vibration of DTX, confirms loading of DTX on GO.63,64 The close proximity of the drug molecules and GO sheets was verified by comparing the fluorescence spectra of GO-DTX with the same concentration of DTX at excitation wavelength of 300 nm. Figure 3c shows that although DTX displays a weak fluorescence at emission wavelength of 370 nm, this fluorescence intensity quenches after drug loading, due to the close binding between DTX and GO.


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Figure 3. (a) UV-vis absorption spectra of GO, DTX and GO-DTX, (b) FT-IR spectra of GO, DTX and GO-DTX (c) Fluorescence spectra of GO, DTX , and GO-DTX at the same concentration of DTX at excitation wavelength of 300 nm


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In order to evaluate the morphological changes of GO sheets after DTX loading, the AFM image of GO-DTX were examined. In Figure 4, the AFM image of GO-DTX and height profile of nanosheets demonstrate an increase in the thickness of GO sheets after drug loading.

Figure 4. AFM image of GO-DTX and corresponding AFM height profile

Targeting and Coating of GO-DTX with Tf-PAH. The prepared GO was stable in deionized water because of the existence of hydrophilic functional groups on its surface and its highly


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negative ζ potential of -45 mV which led to the charge repulsion between GO nanoparticles. However, GO nanoparticles could not be well dispersed in PBS and RPMI cell growth medium, probably due to the neutralization of oxygen functional groups by ionic salts and non-specific binding of proteins.17 To use GO for further applications in biomedicine, it was necessary to improve its stability in physiological solutions. In this research, GO was functionalized with poly (allylamine hydrochloride) (PAH), based on the electrostatic interactions between negatively charged GO-DTX particles and positively charged PAH chains. The high water solubility and cationic nature of PAH enhanced stability of prepared nanocarrier in aqueous medium and physiological environments. The free amine groups on PAH also allowed chemical conjugation of Tf as a targeting agent to PAH. PAH was first conjugated to Tf in an EDC/NHS reaction through formation of amide bonds between amine groups of PAH and activated carboxyl groups of Tf, and then the synthesized Tf-PAH coated the surface of GO. Bradford protein assay shows that Tf is conjugated to PAH at 80% (w/w). The FT-IR spectroscopy was used to confirm conjugation of Tf to PAH (Figure 5a). FT-IR spectrum of PAH shows the vibrations of primary amines (N-H) at 3424 cm-1, 3142 cm-1 (stretching vibrations) and 1620 cm-1, 862 cm-1 (bending vibrations) in addition to the C-N stretching vibration at 1172 cm-1. The C-H stretching and bending vibrations of PAH appear at wavelengths of 2923 cm-1, 2854 cm-1 and 1486 cm-1, 1400 cm-1. In FT-IR spectrum of Tf, the peaks at 1654 cm-1, 1542 cm-1 and 1242 cm-1, correspond to the amide I, derived from the C=O stretching vibrations of the peptide linkages, amide II, mainly from the in-plane N-H bending and from the C-N stretching vibrations, and amide III, mainly from the C-N stretching and from the N-H bending vibrations, respectively.65 Appearance of three new peaks at 1641 cm-1, 1542


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cm-1 and 1249 cm-1 in spectrum of Tf-PAH which correspond to the three amide bonds of Tf, as well as additional absorption peaks at 872 cm-1, 3405 cm-1, 2923 cm-1 and 2854 cm-1 from the out-plane N-H bending and the C-H stretching vibrations of PAH confirm the conjugation of Tf to PAH. Moreover, significant decrease or disappearance of the N-H bending vibration of PAH at 1620 cm-1 and disappearance of the peak of Tf at 1166 cm-1, which is most probably due to the C-O vibration of carboxylic acid groups, can be observed in the Tf-PAH spectrum. Furthermore, compared to the spectra of Tf, the shifts in characteristic peaks, especially in the wavelength of 1076 cm-1 which is shifted to 1064 cm-1, and in the peaks of amide I and amide III, indicate the effect of this conjugation on vibrations of different bonds. These observations suggest the successful formation of amide bond between carboxyl groups of Tf and amine groups of PAH. In addition to FT-IR spectra, the UV-vis spectra of Tf-PAH provide further evidence of conjugation. As shown in Figure 5b, the main UV characteristic peak of Tf at 280 nm appears in the UV-vis spectra of PAH after conjugation with Tf.


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Figure 5. (a) FT-IR spectra of Tf, PAH and Tf-PAH conjugate, (b) UV-vis absorption spectra of Tf, PAH and Tf-PAH conjugate

After the conjugation reaction, the ζ potential of synthesized Tf-PAH was determined to be +50 mV and Tf-PAH bioconjugate coated the negatively charged GO-DTX nanoparticles. The stability and the particle size of Tf-PAH-(GO-DTX), were dependent on the ratio of added TfPAH to GO-DTX in the reaction mixture. The particle size and ζ potential of Tf-PAH-(GODTX) after modification by different values of Tf-PAH were measured. As shown in Figure 6a,


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the ζ potential and the average particle size of GO nanoparticles increased slightly from -45 mV to -39 mV and from 85 nm to 135 nm, after drug loading. The ζ potential of Tf-PAH-(GO-DTX) was negative at R values (mass ratio of Tf-PAH to GO-DTX) less than 0.08, reflecting incomplete integration. As expected, with the increase of Tf-PAH concentration, the ζ potential of Tf-PAH-(GO-DTX) became positive at R value of 0.3 and finally reached a plateau at +47 mV. Furthermore, binding of Tf-PAH at low R values dramatically increased the average particle size of Tf-PAH-(GO-DTX), while the average particle size reduced with further addition of TfPAH. This observation can be explained by ζ potential of nanoparticles. As the ζ potential approached zero at R=0.3 (+4 mV), noticeable aggregation of nanoparticles was observed because of less electrostatic repulsion between nanoparticle. By increasing positive ζ potential, the average particle size reduced, and finally at R=2.5 the nanocarrier with particle size of 186 nm was obtained. The resulting Tf-PAH-(GO-DTX), was evenly and completely coated with positively charged Tf-PAH and exhibited good colloidal stability for months even under physiological conditions. The positive surface charge of Tf-PAH-(GO-DTX), is also beneficial for the cellular uptake and intracellular trafficking, since the cellular membrane of the cells is negatively charged. In addition to electrostatic stabilization, the stability of this drug-loaded nanocarrier even at high salt concentration is a result of steric stabilization and repulsion of long chains of polymers which covered the surface of GO-DTX. The SEM image of Tf-PAH-(GODTX) in Figure 6b, shows the uniform coating of Tf-PAH on the GO-DTX sheets.


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Figure 6. (a) Zeta potential and particle size of Tf-PAH-(GO-DTX) by addition of different amounts of Tf-PAH to GO-DTX measured by DLS, (b) SEM image of GO-DTX sheets covered by Tf-PAH

Drug Release from the Surface of GO in Tf-PAH-(GO-DTX). The release behavior of DTX was studied at pH values of 7.4 and 5.5 over a time period of 133 hours. It was found that DTX showed a pH dependent and sustained release from the surface of Tf-PAH-(GO-DTX), which are in close agreement with others’ reported drug release behavior from GO-based nanostructures.17-


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As shown in Figure 7, only 12.6% of the total loaded DTX was released after 133 hours in

neutral conditions, while the released DTX was much higher within the same time in acidic environment. The DTX was released relatively fast in the first 10-30 hours at pH 5.5 with 21% released drug, but the release rate gradually decreased and finally after 130 hours 34.4% of the DTX was released. This prolonged and biphasic release pattern can be observed in other DTXloaded nanocarriers (Table 1), which is mainly due to the hydrophobic nature of DTX.51-54,56,58-60 The faster release rate in acidic pH is also reported in the DTX-loaded carbon nanotubes.59,60 The slower release rate of DTX from surface of GO particularly at pH 7.4, compared to the other nanocarriers in Table 1, can be explained by surface coating of GO-DTX by Tf-PAH which provides an additional barrier for diffusion of DTX molecules. The strong π-π stacking between DTX and GO sheets as well as possibility of formation of hydrogen bonds between DTX and TfPAH would be other reasons for this sustained release behavior. The higher release rate at acidic conditions can be attributed to the increased protonation of functional groups, which leads to the dissociation of hydrogen bonds between –OH and –COOH groups of GO with -NH and oxygen containing groups of DTX. Furthermore, the weaker electrostatic interaction between GO sheets and Tf-PAH due to the less negative charge of GO sheets, and protonation of COOH groups on GO surface under acidic conditions facilitates diffusion and drug release. This sustained and pH dependent drug release mechanism is desirable for therapeutic applications, since the extracellular tumor tissues and intracellular lysosomes and endosomes are in acidic environment while the body fluid is in neutral conditions.18 The release rate of DTX may be controlled by the amount of PAH loaded on the surface of GO. Moreover, when faster release of DTX is desirable, the release rate of DTX can be increased by covalent attachment of Tf-PAH to GO-DTX, while keeping its stability, instead of physical electrostatic


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interaction and surface coating of GO. This new structure might facilitate DTX penetration through polymer chains, which leads to the faster release of DTX.

Figure 7. Release of DTX from Tf-PAH-(GO-DTX) at two different pH values

Cytotoxic Effect of the Blank Nanocarrier and DTX-Loaded Nanocarrier on MCF-7 Cells. The cytotoxicity of drug loaded nanocarrier on MCF-7 cells was evaluated by morphology of the cells observed under light microscopy. The considerable change in the morphology of MCF-7 cells after incubation with Tf-PAH-(GO-DTX) for 72h demonstrated its cytotoxic effect (Figure 8a). To evaluate the cytotoxicity of DTX-loaded nanocarrier, the blank nanocarrier without drug loading (Tf-PAH-(GO)) was incubated on MCF-7 cell line. As shown in Figure 8b, Tf-PAH(GO) shows cell viability of over 90% at concentration at of 100 µg/ml after 48h and 72h incubation, indicating that it is not cytotoxic. In this study, the concentration of nanocarrier used in all experiments was lower than 100 µg/ml.


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The targeting effect and cytotoxicity of targeted DTX-loaded nanocarrier were compared with the non-targeted one and free DTX, at drug concentrations of 0.03, 0.8, 3.5 and 8 µg/ml. The MCF-7 cells were incubated with the preparations for 72h. Figure 8c, shows a decrease in the relative cell viability of MCF-7 cells treated with free DTX by increasing DTX concentration from 0.03 to 3.5 µg/ml. The poor solubility of DTX in cell medium limited its cytotoxic effect at high drug concentrations, so that a decline in cytotoxicity of DTX was observed at drug concentrations starting at 8 µg/ml. Interestingly, the percentage of cell viability at DTX concentration of 8 µg/ml significantly (P

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