Characterization and In Vitro Sustained Release of Silibinin from pH Responsive Carbon Nanotube-Based Drug Delivery System

1 Materials Synthesis and Characterization Laboratory, Institute of Advanced Technology, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia 2 Laboratory of Vaccine and Immunotherapeutics, Institute of Bioscience, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia 3 Department of Human Anatomy, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia


Introduction
There are various definitions currently circulating when it comes to the term nanotechnology.The prefix "nano" is actually originated from the Greek word "nanos" for dwarf and technology refers to the application of science in a particular subject [1].One nanometer (nm) is equal to onebillionth of a meter and a meter is approximately 39 inches long.According to the declaration made by the United States National Nanotechnology Initiative (NNI), nanotechnology involves research and development at the atomic, molecular, or macromolecular levels at dimensions between 1 and 100 nm to create structures, devices, and systems that are enabled for novel applications.
In recent years, nanobiotechnology (a combination of nanotechnology and biotechnology) especially in medicine, or the so-called nanomedicine, has emerged as one of the most advanced and promising areas.It is extensively used by the academician, particularly in the field of drug delivery.The application of nanotechnology in drug delivery involves the use of carriers and therapeutic agents.Nanoscale drug carriers can significantly enhance the bioavailability and therapeutic efficacy of drugs with reduced side effects.In general, they can be grouped into five categories based on the materials used: polymers, biologics, carbon-based materials, silicon-based materials, or metals [2].Therapeutic agents are drugs or biologically active materials (e.g., nucleic acids and proteins) which can be entrapped, intercalated, encapsulated, dissolved, adsorbed, or attached onto the drug carriers which can then be tailored for controlled and sustained-release formulations [3][4][5].
Carbon nanotubes (CNTs) are carbon-based nanoparticles having rolled-up graphene sheets held together by strong interactions of van der Waals to form large aggregates [6].A single rolled layer of graphite cylinder with a diameter of 0.4 and 2 nm is known as single-walled carbon nanotubes (SWCNTs) [7], whereas multiple concentric cylinders form multiwalled carbon nanotubes (MWCNTs) with a tube diameter ranging from 2 to 100 nm [8].Both nanotubes have unique electrical, optical, thermal, physical, and chemical properties [9] for the development of advanced nanodrug delivery in therapeutic applications.They possess a very high aspect ratio (L/D of 1 : 1000), allowing multiple loading of various biomolecules to be functionalized on the sidewalls of the CNTs via noncovalent adsorption or covalent conjugation [10].The chemical modification (functionalization) of CNTs can help to enhance the biocompatibility and solubility for efficient drug delivery because raw CNTs (nonfunctionalized) are water-insoluble and cannot disperse uniformly in aqueous environment.Recent studies have reported that functionalized CNTs can passively cross cell membranes and deliver attached drugs or bioactives through the enhanced permeability and retention effect without causing any harm to the targeted cells [11,12].
There has been significant progress in the research of functionalized CNTs for the advanced delivery of drugs and biomolecules in biology and nanomedicine [13].A multitude of strategies have been exploited to deliver anticancer drugs (e.g., topoisomerase inhibitors, antimicrotubules antimetabolites, and platinum-based drugs), nonanticancer drugs (e.g., anti-inflammatories, antioxidants, antimicrobials, and antihypertensives), and biomolecules such as antibodies [14], small interference ribonucleic acids (siRNA) [15], and peptide-based vaccines [16] for controlled and targeted drug delivery.Therefore, the emerging field of CNT-mediated drug delivery system no doubt can be expected to generate promising outcomes for delivery of a variety of therapeutic agents.
Silibinin (Figure 1), also known as silybin, is the major polyphenolic flavonoid extracted from the milk thistle Silybum marianum and has been used traditionally as a liverprotective remedy for over 2000 years.Silibinin (SB) can be considered as a safe dietary supplement because it exhibits very low toxicity [17] in humans and animals, possibly due to its antioxidant property [18].In recent years, studies have suggested that SB possess antiproliferative and anticarcinogenic effects in various cancer cell lines, including skin, colon, prostate, ovarian, and bladder cancers [19][20][21].This implicates that SB is a potential drug to treat illnesses related to different types of cancer.However, its effectiveness has been extremely restricted due to its poor aqueous solubility [22] and resulting in poor oral bioavailability, that is, 23-47% [23], after administration.Thus, large dose of SB is required to achieve therapeutic plasma levels.In meeting this demand, a variety of delivery systems have been developed by researches around the globe in order to improve its solubility and thereby bioavailability.For example, formulations using stealth solid lipid nanoparticles [24], mixed micelles [25], microemulsion [26], nanosuspensions [27], porous silica nanoparticles [28], liposomes [23], and nano/microhydrogel matrices [29] as drug carriers have been extensively employed for the enhancement of bioavailability of the poorly watersoluble drug SB.The present study therefore aimed at developing a simple strategy for the conjugation between SB and MWCNTs, secondly, investigating the drug release profile of SB from the CNT-mediated drug delivery system, and thirdly, assessing the cytotoxicity characteristics of MWCNTs functionalized with SB towards human cancer cells (HepG2 and A549).

Materials and Methods
Commercially available multiwalled carbon nanotubes (MWCNTs) were purchased from Chengdu Organic Chemicals Co., Ltd., Chinese Academy of Sciences (Chengdu, China).They consist of short carboxyl carbon nanotubes, where some -COOH groups have been grafted onto the surface of CNTs.Detailed information for the purchased CNTs is included in Table 1.The commercial silibinin (>98% pure as checked by HPLC analysis) was purchased from Sigma-Aldrich (Buchs, Switzerland).HepG2 (human liver hepatocellular carcinoma cell line), and A549 (human lung adenocarcinoma epithelial cell line) were purchased from ATCC (USA).All the other chemicals and solvents were of analytical grade.Purified water was produced by a Millipore water purification system.

Conjugation of MWCNTs with SB. SB was conjugated to
the COOH-MWCNTs by a simple method without using any cross-linker agent.10 mg of COOH-MWCNTs was dispersed in a 5 mL of ethanol followed by sonication in a water bath for 1 h at room temperature and later added to SB solution (2 mg of SB fully dissolved in 40 mL of 99.8% ethanol).The mixture was then stirred continuously using magnetic stirrer for 20 h at room temperature in darkness.This is to prevent degradation of the drug.Subsequently, the final product (SB-MWCNTs) was collected by filtration, repeated centrifugation, and washing with deionized water to remove excessive SB.Finally, the sample was dried in an oven at 60 ∘ C overnight.To determine the amount of unbound SB, the absorbance of the centrifuged solution and free SB were measured at wavelength of 288 nm, which is the characteristic absorbance of SB (Figure 2).

Physicochemical Characterization of SB-MWCNTs.
The drug loading capacity and controlled-release profiles of the nanohybrid were determined by a Lambda 35, ultravioletvisible spectrophotometer (Perkin Elmer) at a wavelength of 288 nm.The drug loading capacity was calculated using the following formula: The drug loading capacity was estimated to be ∼ 35.1%.The formation of SB-MWCNTs nanohybrid was confirmed by studying the characteristic absorption bands associated with functional groups of -COOH and SB molecules using Fourier transform infrared (FTIR) spectroscopy.The samples were recorded at wavelengths ranging from 500 to 4000 cm −1 on a Thermo Nicolet Nexus FTIR spectrophotometer (model Smart Orbit).For FTIR measurement, the samples were prepared and mixed with potassium bromide (KBr) powder and formed into pellets.Raman spectroscopy measurements were performed on powdered samples using a RamanMicro 200 (Perkin Elmer) instrument at an excitation wavelength of 785 nm.Thermogravimetric studies were performed on a TGA Q500 (TA instruments) to determine the actual drug loading.The samples were heated from 25 to 1000 ∘ C with a heating rate of 10 ∘ C/min under a nitrogen purge of 40 mL/min.
The surface morphology of the sample was analyzed via scanning electron microscope (SEM).The sample was placed on a stub and sputter coated with gold and examined at 5 kV in a JEOL JSM-7600F field emission scanning electron microscope.Transmission electron microscope (TEM) micrographs were obtained using a Hitachi H-7100 by placing a droplet of sonicated dispersion of the sample directly on the carbon-coated copper grid and dried at 37 ∘ C for 24 h.

In Vitro Drug Release Studies.
The drug release experiment from SB-MWCNTs was studied on different human body simulated pH 7.4 and 4.8 phosphate-buffered saline (PBS) solutions.About 11.43 mg of the nanohybrid sample was added to 40 mL of the PBS solutions.The amount of SB released in the solution was measured at predetermined time intervals using a UV-Vis spectrophotometer at 288 nm.

Cell Lines and Culture
Conditions.HepG2 and A549 cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and cultivated as adherent monolayers in T75 flasks at 37 ∘ C in a humidified atmosphere of 5% CO 2 .Cells were subcultured (i.e., at approximately 80% confluence) using 0.25% trypsin-EDTA solution for all the experiments.

Determination of Cytotoxicity (MTT Assay).
For the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, all cancer cells were seeded at a density of 1 × 10 4 cells per well in 100 L of culture medium in 96-well plates and incubated at 37 ∘ C in a 5% CO 2 atmosphere for 24 hours before being used in cell viability assays.The SB compound, COOH-MWCNTs, and nanohybrid were prepared at various concentrations, and the treated cells were incubated for 72 hours.Subsequently, 20 L of freshly prepared MTT solution was added to each well and culture plates were incubated at 37 ∘ C for 3 hours until a purple-colored formazan product developed.The solution in each well, containing media, unbound MTT, and dead cells, was removed and 100 L of dimethyl sulfoxide (DMSO) was added to each well and the solution was vigorously mixed to dissolve the reacted dye.The absorbance of each well was read on a microplate reader at a test wavelength of 570 nm.All assays were done with three parallel samples in triplicate independently.The cytotoxicity of the free drug, COOH-MWCNTs, and nanohybrid were expressed as the percentage of cell viability with respect to control cells.2.6.Statistical Analysis.Statistical analysis was performed using the statistical package for social science (SPSS) version 20.0.Results were analysed by one-way analysis of variance (ANOVA).Data were expressed as mean ± standard deviation (mean ± SD).A difference was considered to be significant at  < 0.05.

Fourier Transform Infrared Spectroscopy.
To determine the presence of reactive groups on the COOH-MWCNTs surface after conjugation of SB molecules, Fourier transform infrared spectroscopy (FTIR) was used.The FTIR spectra of commercially purchased COOH-MWCNTs, SB, and SB-MWCNTs nanohybrid are shown in Figure 3.The peak at 3436 and 1700 cm −1 (Figure 3(a)) is associated with the presence of hydroxyl group (-OH) and the C=O stretching of the carboxylic acid group (-COOH), respectively.Similar FTIR observation was also reported by a group of researchers for their work on the synthesis of carboxylated MWCNTs [30].Another peak at around 1570 cm −1 of the nanotubes corresponds to C=C stretching vibration in the carbon skeleton [31].Figure 3(b) shows the spectrum of the pure SB where the characteristic absorption due to the -OH stretching vibration occurs at 3452 cm −1 [32].When the COOH-MWCNTs and drug interact, the FTIR spectra will show broadening of the functional groups and band shift compared to that of the nanocarrier and pure drug.As presented in Figure 3(c), the absorption band was slightly shifted from 1636 cm −1 for the C=O stretching in pure SB to 1630 cm −1 in nanohybrid.Each -COOH moiety of the nanocarrier has C=O and -OH groups that can potentially form hydrogen bonds with SB [33].

Raman Scattering Studies.
Raman spectroscopy is a powerful technique widely used to characterize the structural quality of functionalized CNTs.As shown in Figure 4, the G-and D-bands of COOH-MWCNTs were exhibited at 1665 cm −1 and 1447 cm −1 which were assigned to the inplane E 2 g zone-center mode and disorder-induced mode, respectively.The Raman spectrum of the SB-MWCNTs nanohybrid does not show recognizable D-band and the Gband peak intensity was much weaker and slightly shifted towards lower frequency compared to that of the COOH-MWCNTs.This indicates that the characteristic absorption peaks were strongly attenuated due to the conjugation process [34], hence confirming that the interactions between COOH-MWCNTs and drug are strong enough to change the structural pattern of the nanotubes [35].Furthermore, the radial breathing modes are too weak to be detected due to the large diameters of the nanotubes used in this study.weight loss from the nanocarrier in the temperature range of 125-750 ∘ C is approximately 6.4%, which is the typical weight loss for acid-functionalized MWCNTs [34].The weight loss occurred in the 125 ∘ C to 425 ∘ C region may be attributed to the evaporation of absorbed water as well as decomposition of carboxyl functionalities attached to the MWCNTs walls [36][37][38].Subsequently, the weight loss in the temperature range between 425 ∘ C and 625 ∘ C corresponds to the removal of hydroxyl groups from the surface of nanotubes [39].Finally, the residue at 625 ∘ C and above should be explained by the oxidation of the remaining disordered carbon in the sample [40].In the heating up to 1000 ∘ C, both COOH-MWCNTs and SB-MWCNTs lost about 19.1% and 52.8% of total weight, respectively.Therefore, approximately 34 wt% of the mass loss was due to the decomposition of SB, further confirming that SB molecules have been adsorbed to COOH-MWCNTs.This finding is in agreement with the drug loading capacity determined by ultraviolet-visible spectrophotometer where the percentage of drug loading was calculated to be around 35.1%.

Surface Morphology.
The microstructures of the COOH-MWCNTs as well as the synthesized SB-MWCNTs nanohybrid were studied by FESEM and TEM. Figure 6 presents FESEM images of MWCNTs before (Figure 6(a)) and after drug conjugation (Figure 6(b)).As seen in Figure 6(a), the nanotubes have a short, tubular topology with smooth surface and they are held together into bundles [30] while Figure 6(b) demonstrates the rougher surface of the MWCNTs and they appear to be in a slightly dispersed form.Similar observation has been reported in the literature for related drug-MWCNTs experiment [41].was very rapid at the first 100 minutes when exposed to pH 7.4 and pH 4.8 solutions, respectively.Subsequently, the release of SB became much slower and sustained, with total release equilibrium of approximately 96.6% within 1000 minutes at pH 7.4 and 43.1% within 800 minutes at pH 4.8.This process may be attributed to the different extents of COOH ionizations in aqueous solution of different pH values, indicating that the release rate of SB from the nanocarrier is pH-dependent.It was also observed that the SB-MWCNTs nanohybrid has a higher release profile when exposed to pH 7.4 compared to pH 4.8.This is because the higher the pH values, the more carboxylate anions would be produced to form homogenous solution [42], giving rise to a higher solubility of the nanocarrier.
To determine the release kinetics of SB from the nanocarrier, three kinetic models, namely, pseudo-first order equation (2), pseudo-second order equation (3), and parabolic diffusion equation (4), were used [43][44][45]: where   and   refer to the equilibrium release amounts at time ,  is a constant corresponding to release amount, and  0 and   represent the drug content remaining in SB-MWCNTs at release time 0 and , respectively, and  is a constant.
By fitting the control release data of SB from MWCNTs nanocarrier using the above kinetic models, it was observed that the pseudo-second order kinetic model was best to describe the release kinetic behaviour of SB from the MWC-NTs nanocarrier.Figure 9 presents the plot of the fitting of SB released from MWCNTs nanocarrier and the corresponding correlation coefficient ( 2 ) and  values are shown in Table 2.At pH 7.4, both  2 and  values are 0.9980 and 1.82 × 10 −4 mg/min, respectively, and at pH 4.8, the corresponding values are 0.9955 and 4.13 × 10 −4 mg/min, respectively.

In Vitro Anticancer Effect of SB-MWCNTs.
In order to evaluate the potential application of the synthesized nanohybrid in nanomedicine and to understand their cytotoxicity nature, in vitro cytotoxicity tests were conducted on two human cancer cell lines (HepG2 cells and A549 cells).The treatment time was 72 h.The cell viability was examined by MTT assay (Figure 10) at different gradient concentrations of 0.78, 1.56, 3.13, 6.25, 12.50, 25, and 50 g/mL.
Since SB has demonstrated its carcinogenic potential for the treatment of cancer [46,47], we hereby investigated the response of two human cancer cell lines, that is, HepG2 (human liver cancer cells) and A549 (human lung cancer cells).Based on the results presented in Figures 10(a) and 10(b), SB-MWCNTs nanohybrid significantly inhibited proliferation of cancer cells especially A549 cells, at lower concentrations of 1.56-6.25 g/mL when compared to free drug alone.Although the same concentrations of SB-MWCNTs and free drug are compared, the amount of drug loaded onto MWCNTs (34% wt as determined by TGA analysis) is actually much lower than the amount of free drug used in the assay.Therefore, this sustained release formulation can be used to enhance the efficiency of cancer therapy with reduced drug-related side effects.In addition, further cellular and molecular investigations will be necessary to examine its cytotoxic nature in drug delivery and controlled release applications.

Conclusions
The SB molecules were conjugated to MWCNTs by a simple method based on commercially available carboxylated MWCNTs.The chemical conjugation between COOH-MWCNTs and SB molecules has been confirmed by FTIR spectroscopy, Raman, and TGA studies.The FTIR results Raman spectroscopy and TGA analysis provided further support for the success of chemical conjugation between nanocarrier and drug.Release of the drug from the nanocarrier was observed to occur in a sustained-and pHdependent manner, suggesting that this nanohybrid could be developed as a sustained-and controlled-release formulation for SB.It was observed that the release kinetic behaviour of SB from the MWCNTs nanocarrier conformed well to pseudosecond order.Proliferation assay against cancer cells indicates that SB-MWCNTs expressed cytotoxicity at lower dosages in comparison with free drug.These preliminary results justify further development of this formulation for drug delivery in nanomedicine.

Figure 7 (
a) illustrates the typical TEM image of COOH-MWCNTs in which the nanotubes are seen bundled together as observed in FESEM (Figure 6(a)) and Figure 7(b) shows more exfoliated nanotubes after drug conjugation.3.5.In Vitro Drug Release Study.The cumulative release profile of SB from MWCNTs in PBS solutions is presented in Figure 8.The amount of SB released from the MWCNTs

Figure 8 :
Figure 8: Release profiles of silibinin from MWCNTs at pH 7.4 and pH 4.8 up to 1000 minutes.

Figure 9 :Figure 10 :
Figure 9: Fitting data of SB from MWCNTs nanocarrier into PBS solutions at pH 7.4 and pH 4.8 using pseudo-first and pseudo-second order kinetics and parabolic diffusion model.The release of kinetic behaviour of SB from its nanocarrier conformed well to pseudo-second order.

Table 1 :
The properties of the commercial multiwalled carbon nanotubes provided by the manufacturer.

Table 2 :
Correlation coefficient ( 2 ) and rate constants () obtained by fitting the silibinin release data for the COOH-MWCNTs in phosphate-buffered saline solutions at pH 7.4 and pH 4.8.