Nano-based materials or nanotechnology research has become a hot zone of material science research globally, due to the various advantages that come with nanomaterials [1
], especially in numerous biomedical applications [2
]. Chemotherapy remains the best choice of cancer treatment, due the availability of various anticancer agents. Nevertheless, the challenge of toxicities posed by these agents still exits [7
]. Over a decade or so, various nanotechnology platforms have been explored in overcoming this challenge. Some of the areas of interest are the nanodrug delivery systems (NDDS). However, multimodal delivery systems (MDS) are recently gaining more attention [8
], where drugs are simultaneously loaded along with other active agents on the same nanocarrier platform [7
]. For instance, in theranostic research, the concept of MDS is employed where a nanocarrier is used as a delivery agent for therapeutic agents and diagnostic agents, such as magnetic resonance imaging (MRI) contrast agents [6
], for a non-invasive concurrent delivery [10
MRI is a non-invasive and non–ionizing important imaging tool used for cancer clinical diagnosis. Since the 1970s, MRI has been one of the most recognized powerful imaging techniques due to its high spatial resolution and tissue penetrating ability [8
]. However, the use of MRI often requires contrast agents. Gadolinium-based (Gd) contrast agents are the commonly used contrast agents for MRI, which improve the T1 and T2 relaxation times of the images produced [8
]. T1 and T2 relaxations are the main signals among others generated during MR imaging, which have distinctive grey-scale color contrasts reflecting the fluid and soft tissue composition of human subject. The signals represent spin–lattice relaxation and spin–spin relaxation for T1 and T2, respectively [12
]. Zn/Al-layered double hydroxide (LDH) is one of the candidates capable of simultaneously intercalating and adsorbing theranostic agents due to their exchangeable interlayer anions. LDH is one of the group of two-dimensional layered structures materials [13
] and has the general formula of [
], where divalent and trivalent metal cations are represented by M2+
, respectively, and interlayer exchangeable anions are represented by [An−
], and water as x
]. Gallic acid is the therapeutic agent employed in this research; it is a naturally occurring polyhydroxyl phenolic compound, often found in different kinds of fruits. It is believed to have anticancer properties as well as other activities in a range of cells [18
]. Although there are various research publications on drug intercalation using LDH in drug delivery as reviewed by Kura et al. [19
], only a few works have so far been done on theranostic applications using LDH-based nanocarriers. Those articles have also been reviewed by Usman et al. [8
], amongst which none has reported synthesis of theranostic nanocomposite using drug intercalation process.
Herein, we synthesized theranostic nanoparticles by Gd doping onto Zn/Al-LDH. Gallic acid was first intercalated into the interlayers of the LDH-Gd and AuNPs were then grown on the surface of the LDH nanoparticles. The LDH prepared via co-precipitation method was used as the nanocarrier, while Gd and AuNPs were used as the main contrast agent and booster for MRI, respectively.
3. Materials and Methods
Gallic acid with molecular weight of 170.12 g/mol and 98% purity, sodium hydroxide molecular weight of 40.00 g/mol and 98% purity and phosphate-buffered saline were purchased from Sigma-Aldrich (St. Louis, MO, USA). Zinc nitrate hexahydrate molecular weight of 297.47 g/mol, 98% purity, and aluminium nitrate hexahydrate molecular weight of 375.13 g/mol, 98% purity, were purchased from Systerm ChemPur (Shah Alam, Selangor Darul Ehsan, Malaysia). Gadolinium (III) nitrate hexahydrate and molecular weight of 451.4 g/mol with 99.9% purity and tetrachloroauric(III) acid trihydrate, 393.83 g/mol and 49% Au purity were purchased from Acros Organics (Morris Plains, NJ, USA). Sodium borohydride molecular weight of 37.83 g/mol, and 99% purity was purchased from Fluka Analytical (St. Gallen, Switzerland). All chemicals were used as received without further purification. Deionized water was used throughout the experiment.
3.2. Synthesis of Gd-Zn/Al-Layered Double Hydroxide
Zn/Al-layered double hydroxide was synthesized using Zn(NO3)2 and Al(NO3)3 at a molar ratio of 4:1 of to . Gd(NO3)3 (0.0008 M) was firstly dissolved in 250 mL deionized water before the addition of the Zn/Al nitrate salts. Dropwise addition of NaOH (2 M) was immediately followed until a pH of 7 was reached. The synthesis was conducted under nitrogen flow and vigorous stirring. The slurry obtained at the end of the process was aged for 18 h at 70 °C, centrifuged, washed with deionized water (three times), and oven dried at 60 °C.
3.3. Loading of Gallic Acid into Gd-Zn/Al-LDH Nanoparticles (GAGZA)
Briefly, gallic acid, 0.2 M solution was prepared by dissolving 3.6 g of the drug into 50 mL deionized water while stirring and heating at 45 °C. Under continuous nitrogen flow with vigorous stirring, the drug solution was simultaneously added dropwise with NaOH (2 M) into the 250 mL solution of Gd-Zn/Al-layered double hydroxide. The solution was kept until pH 7 was reached and the drug was completely loaded. The mixture was then aged for 18 h at approximately 70 °C. The resultant slurry obtained was filtered, washed/centrifuged using deionized water, and oven dried at 60 °C for 12 h.
3.4. Doping of Gold Nanoparticles (AuNPs) onto Zn/Al-Gd GA LDH (GAGZAu)
Appropriate amount of GAGZA was ultrasonically dispersed for 5 min, under gentle stirring; 2% HAuCl4 (6 mL) solution was added. After 5 min stirring, 0.125 M NaOH (4 mL) was added; the dispersion was allowed to stir for 5 min before the temperature of the solution was raised to 60 °C and stirred for 24 h in dark conditions. The resulting precipitate was obtained after centrifuge/filtration, the mixture was re-dispersed in 30 mL deionized water; 1 M NaBH4 (20 mL) was added and stirred for an hour. The final suspension obtained was washed six times using a centrifuge, filtered, and dried at 70 °C in an oven.
An XRD-6000 (Shimadzu, Tokyo, Japan) X-ray diffraction (XRD) instrument was used for crystallographic analysis of the powdered samples. CuKα radiation (λ = 1.5418 Å) at scan speed of 4°/min, using a range of 2–70 °C at 30 kV and 30 mA was used in obtaining the XRD patterns. A ultraviolet–visible (UV–Vis) spectrophotometer (PerkinElmer, Singapore) (Lambda35) was used for the controlled release and optical property studies. Fourier transform infrared (FT-IR) spectra of the materials were obtained using a Thermo Nicolet, Nicolet 6700 model (Thermo Scientific, Waltham, MA, USA). The spectra were obtained using the potassium bromide (KBr) discs at 10 ton pellet pressing and a resolution of over a range of 400–4000 . A PerkinElmer spectrophotometer (PerkinElmer, Wellesley, MA, USA) (model Optima2000DV) inductively coupled plasma atomic emission spectrometry (ICP-ES) was employed in studying the composition of zinc, aluminum, gadolinium, and gold content of the nanoparticles. A CHNS-932 LECO (LECO, St. Joseph, Michigan, USA) instrument was used in determining carbon, hydrogen, nitrogen, and sulphur (CHNS) content in the sample. A Mettler-Toledo instrument (METTLER TOLEDO, Shah Alam, Selangor, Malaysia) was used for thermogravimetric and differential thermogravimetric (TGA/DTG) analyses. A sample heating rate of 10 °C/min and a range of 20–1000 °C were used in this work. The analysis was carried out at a continuous nitrogen flow at 50 mL/min flow rate. High resolution transmission electron microscope (Hi-TEM) (FEI Tecnai TF20 X-Twin, Hillsboro, OR, USA) and FEI Nova NanoSEM 230 field emission scanning electron microscope (FESEM) (FEI, Hillsboro, OR, USA) were employed for shape/sizes analyses and morphological analyses, respectively while energy dispersive X-ray spectroscopy (EDX) (FEI, Hillsboro, OR, USA) was used for elemental analysis of the FESEM micrographs.
3.6. Drug Release Study
As mentioned earlier, gallic acid release from GAGZA was studied using a PerkinElmer Lambda 35 UV-Vis spectrophotometer (PerkinElmer, Singapore). Firstly, standard solutions of gallic acid at different concentrations were prepared. Appropriate amount of GAGZA was dissolved in 5 mL of HCl (1 mol/L) and then diluted with 45 mL deionized water. The lambda max of the drug in the solution was found to be 264 nm, which was used in determining the standard curve for the standard solutions and drug loading capacity of GA. Kinetic release of the loaded GA from GAGZA was done at pH 7.4 and 4.8 in a phosphate-buffered solution (PBS). Briefly, 25 mg of the sample was dispersed in 30 mL of the PBS in tubes. The tubes were placed in an oil bath shaker at 37 °C and 3 mL of the solutions were withdrawn and replaced with 3 mL of pure PBS at time intervals of 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 12.0, 24.0, 48.0, 72.0, 92.0, and 120.0 h. The release media extracted were analyzed at lambda max = 264 nm wavelength with a UV–Vis spectrophotometer (PerkinElmer, Singapore).
3.7. Cell Culture
The cell lines used for cancer study was HepG2 (human liver hepatocellular carcinoma cell line) and normal cell lines, 3T3 (standard fibroblast cell line) for toxicity study, which were obtained from ATCC. RPMI 1640 was used as a medium for cell lines growth, which contains 1% penicillin/streptomycin and 10% fetal bovine serum (FBS). Cells culture was done at approximately 80% confluence, as adherent monolayers. Temperature was set at 37 °C and in a 5% humidified atmosphere. Cell harvest was done via trypsinization (in brief) with trypsin-EDTA solution. All reagents are research grade and were used as received.
3.8. MTT Cell Viability Assays
RPMI 1640 was also used as a medium for the cancer cell lines and normal cells, which were grown in a humidified incubator at 37 °C and 5% . The cells were grown and were harvested and counted. Prior to 24 h incubation, the cells were transferred to 96-well plates (1 × 104 cells/well) and then the GAGZAu nanoparticles, LDH, and free GA were added. The medium was kept for 24 h to allow the cells to attach to the surface before treatment. The cells containing the GAGZAu, Zn/Al-LDH, and free drug were administered and applied in different concentrations prior to treatment and 72 h incubation. For MTT, 5 mg of (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide was dissolved in PBS (2 mL). To each of the 96-well plates, 20 μL of the MTT solution was added and incubated at 37 °C for 3 h until formazan product was developed (purple-colored). Suction method was employed to remove the solution in each well containing media, unbound MTT, and dead cells; 100 μL of DMSO was added to each well. The optical densities of the cells were obtained with the use of a microplate reader at 570 nm. Prior to the measurements, the cells were shaken. All analyses were done in triplicate and the cell viabilities/increase was presented in percentage in reference to control cells.
3.9. MR Imaging Analysis
The GAGZAu nanocomposite was tested for MRI signal intensity using 3.0 T MRI clinical instrument (3.0 T Siemens Magnetom, Erlangen, Germany). Prior to the analysis, GAGZAu was prepared in various concentrations (2.0, 0.5, and 0.2 w/v) according concentration. Gd(NO3)3 (0.5 w/v) and water together with samples were then placed in a 1 mL tube. The MR image was acquired by attaching the tubes to an MRI phantom, and then the phantom was placed in the instrument. The T1-weighted images of the samples were captured at TR/TE: (83/9000) 224 × 220 s, field of view (FOV): 120 × 120. The MR image was analyzed and signal intensities of the individual samples were extracted with Syngovia (MRI and CT reporting software, syngo MR E11, Siemens, Erlangen, Germany, 2013).