Improved Anticancer Effect of Magnetite Nanocomposite Formulation of GALLIC Acid (Fe3O4-PEG-GA) Against Lung, Breast and Colon Cancer Cells

Lung cancer, breast cancer and colorectal cancer are the most prevalent fatal types of cancers globally. Gallic acid (3,4,5-trihydroxybenzoic acid) is a bioactive compound found in plants and foods, such as white tea, witch hazel and it has been reported to possess anticancer, antioxidant and anti-inflammatory properties. In this study we have redesigned our previously reported anticancer nanocomposite formulation with improved drug loading based on iron oxide magnetite nanoparticles coated with polyethylene glycol and loaded with anticancer drug gallic acid (Fe3O4-PEG-GA). The in vitro release profile and percentage drug loading were found to be better than our previously reported formulation. The anticancer activity of pure gallic acid (GA), empty carrier (Fe3O4-PEG) nanocarrier and of anticancer nanocomposite (Fe3O4-PEG-GA) were screened against human lung cancer cells (A549), human breast cancer cells (MCF-7), human colon cancer cells (HT-29) and normal fibroblast cells (3T3) after incubation of 24, 48 and 72 h using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT assay. The designed formulation (Fe3O4-PEG-GA) showed better anticancer activity than free gallic acid (GA). The results of the in vitro studies are highly encouraging to conduct the in vivo studies.


Introduction
Cancer is one of the serious concerns around the world and it is one of the main causes of death worldwide [1]. In a press release by the World Health Organization in 2014, it was reported that cancer accounted for 8.2 million deaths worldwide in 2012 with lung, breast and colorectal cancers identified as most common occurrences worldwide in the year 2012 [2]. This was further supported by a press release by the World Health Organization in 2015. According to the Cancer statistics 2017, in the United States alone, about 1,688,780 new cancer cases and 600,920 cancer deaths are projected to occur [3]. Current cancer treatments rely on radiation and chemotherapeutic agents that work by killing rapidly dividing cells in the body. The main drawback of conventional chemotherapy is the adverse effects on the body as it cannot deliver selective action specifically to the cancer cells, thus damaging the surrounding normal healthy cells or rapidly dividing healthy cells such as the cells of gastrointestinal tract, bone marrow, hair follicles, causing issues like cardiac, hepatic, pulmonary, renal and gastrointestinal toxicities [4][5][6]. Drug delivery systems offer numerous advantages over tradition chemotherapy such as targeted delivery at disease site, sustained release leading to prolonged bioavailability, lower dosage requirement and improved drug solubility among others [7][8][9][10][11][12]. Significant impact has been made by the application of nanotechnology in medicine for theranostic agents development which can diagnose and cure the diseases simultaneously [13,14]. Variety of nanocarriers have been designed and successfully applied for the delivery of the therapeutic agents such as graphene oxide, polymers-based delivery systems, layered double hydroxides, gold nanoparticles, multifunctional nanoparticles and iron oxide magnetite nanoparticles [8,[15][16][17][18][19][20][21]. Polymer coated iron oxide magnetite nanoparticles (Fe 3 O 4 ) have received much of the attention as the novel cancer therapeutics vectors because of their unique properties such as ease of preparation, easily scalable production, sustained release properties, high encapsulation capacity, biocompatibility with normal cells and tissues, easier surface modification and stable magnetic nature [9,[22][23][24][25][26]. All of these characteristics make iron oxide magnetite nanoparticles (Fe 3 O 4 ) an ideal candidate for cancer delivery vectors. However, the large surface area to volume ratio and the dipole-dipole attraction causes the agglomeration of nanoparticles, hence the need for surface polymer modification. The magnetic nanocarriers need to be stable in normal saline and water at neutral pH for biological, medical diagnostic and therapeutic applications [23,27]. To avoid agglomeration, the surface of iron oxide magnetite nanoparticles is coated with polymer which also helps in sustained release and better stability in physiological conditions. The polymer poly (ethylene-glycol) (PEG) has been widely applied in drug delivery and is being utilized as protective layer for the nanoparticles. The monomer unit of PEG contains both polar oxygen and two methylene group which are non-polar. This dual polarity makes PEG to be soluble in variety of polar and non-polar solvents and has been widely used to improve aqueous solubility of hydrophobic drugs [28,29]. Gallic acid (3,4,5-trihydroxybenzoic acid) is a bioactive compound found in plants and foods such as white tea, witch hazel and it has been reported to possess antioxidant, anti-inflammatory, anticancer properties and is also known for its protective activity on normal cells which makes them pivotal for cancer therapy [30]. In this study we have redesigned an anticancer nanocomposite formulation of Gallic acid loaded on iron oxide magnetite nanoparticles coated with polyethylene glycol (Fe 3 O 4 -PEG-GA) with improved drug loading and better sustained release properties and was characterized by X-ray diffraction (XRD), dynamic light scattering (DLS), in vitro cytotoxicity assay and drug loading quantification. In previous study we tested Gallic acid nanocomposite formulation [(P-Fe 3 O 4 -PEG-GA) (in formula P stands for previous)] against MCF-7, a breast cancer cell line with the IC 50 value of 11.61 ± 0.12 µg/mL and human normal lung fibroblast cells MRC-5 was used as a model for normal cell in which more than 80% cell viability was observed after 72 h incubation [31]. In this study we tested the free drug GA, empty nanocarrier and the anticancer nanocomposite formulation Fe 3 O 4 -PEG-GA against A549 human lung carcinoma cells, HT29 human colon adenocarcinoma cell line, repeated on MCF-7 breast cancer cells and normal 3T3 cells for incubation period of 24, 48 and 72 h.

Dynamic Light Scattering (DLS)
The size of the anticancer nanocomposite Fe3O4-PEG-GA was determined using Zetasizer by dynamic light scattering (DLS). The sample was dispersed in water and sonicated for 15 min and then analyzed with Zetasizer. The sample was found to have narrow size distribution between 5 and 12 nm with average particle size of 10 nm as shown in Figure 1b. This size distribution is much smaller compared to previously reported (P-Fe3O4-PEG-GA) which had wide distribution 20-50 nm with average size of 31.44 nm [31].

In vitro Release Studies
Release behavior of GA from the nanocomposite Fe3O4-PEG-GA was conducted in human body simulated buffer saline (PBS) solution of pH 7.4 (human blood pH) and in pH 4.8 (intracellular lysosomal pH) as shown in Figure 1c. For the release studies 10 mg of the nanocomposite was put in 10 mL PBS solution of pH 7.4 and pH 4.8 in thermostat at 37 °C with constant shaking. At different

Dynamic Light Scattering (DLS)
The size of the anticancer nanocomposite Fe 3 O 4 -PEG-GA was determined using Zetasizer by dynamic light scattering (DLS). The sample was dispersed in water and sonicated for 15 min and then analyzed with Zetasizer. The sample was found to have narrow size distribution between 5 and 12 nm with average particle size of 10 nm as shown in Figure 1b. This size distribution is much smaller compared to previously reported (P-Fe 3 O 4 -PEG-GA) which had wide distribution 20-50 nm with average size of 31.44 nm [31].

In vitro Release Studies
Release behavior of GA from the nanocomposite Fe 3 O 4 -PEG-GA was conducted in human body simulated buffer saline (PBS) solution of pH 7.4 (human blood pH) and in pH 4.8 (intracellular lysosomal pH) as shown in Figure 1c. For the release studies 10 mg of the nanocomposite was put in 10 mL PBS solution of pH 7.4 and pH 4.8 in thermostat at 37 • C with constant shaking. At different time points 3 mL aliquot was taken out and replaced with new buffer of either solution of pH 7.4 and pH 4.8 respectively and analyzed for the percentage release using UV-Vis spectrophotometer (Waltham, MA, USA). The release of GA was found to be sustained in both physiological pHs (7.4 and 4.8) and took 200 h (about 8 days) for the complete release in both conditions as shown in Figure 1c. The release profile of GA from the nanocomposite Fe 3 O 4 -PEG-GA is much more sustained compared to previously designed nanocomposite P-Fe 3 O 4 -PEG-GA which initially showed burst release with more than 40% drug released in less than 2 h [31]. Moreover, the release profile of free drug GA has been reported to be extremely fast which took less than 2 min for the complete release [31]. This suggests that nanocomposite designed (Fe 3 O 4 -PEG-GA) has much better sustain release profile compared to free drug and the previously designed nanocomposite (P-Fe 3 O 4 -PEG-GA).

Drug Loading Percentage Quantification
Percentage loading of the GA in the nanocomposite Fe 3 O 4 -PEG-GA was determined with (UV-Vis) spectrophotomer (Waltham, MA, USA) GA acid was extracted (deloaded) from the nanocomposite Fe 3 O 4 -PEG-GA by putting 10 mg of it in 50 mL of 1 molar phosphate buffer saline (PBS) solution, followed by sonication for an hour and kept in thermostat with constant shaking at 37 • C for 10 days. After that sample was filtered and filtrate was subjected to quantification of GA. For quantification different concentrations of GA standards were prepared e.g., 25, 50, 100, 150 and 200 ppm and were analyzed and correlation coefficient (r 2 ) was found to be 0.9918. After that different filtrate was analyzed three times and loading was found to be 35%. The percentage loading of GA in this redesigned nanocomposite is much higher (35%) compared to our previously reported designed nanocomposite of Fe 3 O 4 -PEG-GA in which loading percentage of GA was found to be very lower i.e., 7%. The improved loading can be ascribed to solvent used to dissolve GA i.e., (80% Methanol:20% water).

Anticancer Assays
The free drug GA, empty nanocarrier (Fe 3 O 4 -PEG) and designed magnetite nanocomposite formulation (Fe 3 O 4 -PEG-GA) were tested against different cancer cell line namely lung cancer cell (A549), breast cancer cell (MCF-7) and colon cancer cell (HT-29) lines to determine their anticancer properties. In our previous studies we had tested the nanocomposite (P-Fe 3 O 4 -PEG-GA) against breast cancer cell (MCF-7) in this study we repeated the assay on breast cancer cell (MCF-7) as the percentage drug loading of GA is different than previously reported (P-Fe 3 O 4 -PEG-GA). Doxorubicin, a chemotherapeutic drug, has been studied extensively and its IC 50 values towards the same cancer cell lines have been reported to be 0.33 ± 0.03, 0.05 ± 0.01 and 0.58 ± 0.01 µg/mL for HT-29, MCF-7 and A549 respectively [33].These IC 50 values of Doxorubicin were used as a reference for the positive control in this study. Previous anticancer studies involving A549 used a higher range of concentration of GA as well, ranging from a minimum of 10 µg/mL up to 500 µg/mL [34][35][36]. All the samples with above concentrations were incubated for 24, 48 and 72 h with the A549 lung cancer cells as shown in Figure 2d-f respectively. The designed anticancer nanocomposite Fe 3 O 4 -PEG-GA showed better anticancer activity (IC 50 37.49 µg/mL) compared to free drug GA (IC 50 56.49 µg/mL). The effective IC 50 concentration (i.e., actual amount of GA based on percentage drug loading) present in the nanocomposite 13.12 µg/mL is much lower than IC 50 of whole nanocomposite 37.4 µg/mL. So in reality, effective IC 50 of nanocomposite 13.121 µg/mL compared to IC 50 free GA (i.e., 56.49 µg/mL) much lower against lung cancer cells A549. Table 1 shows the IC 50 of free drug GA, and the nanocomposite Fe 3 O 4 -PEG-GA compared to Doxorubicin.   [31]. The better anticancer activity of nanocomposite can be attributed to the nanosize and sustained release properties of the anticancer nanocomposite. Table 1 shows the IC 50 vales of the nanocomposite and free drug GA against Doxorubicin.

Anticancer Activity against Colon Cancer Cell (HT-29)
The free drug GA, empty nanocarrier and the nanocomposite Fe 3 O 4 -PEG-GA were tested against colon cancer cell (HT-29) using different concentrations i.e., 0.47, 0.94, 1.88, 3.75, 7.5, 15 and 30 µg/mL and incubated for 24, 48 and 72 h as shown in Figure 2j-l respectively. The IC 50 of free drug GA was found to be 15 µg/mL. The IC 50 of the nanocomposite Fe 3 O 4 -PEG-GA was found 4.85 µg/mL and the effective IC 50 of the nanocomposite Fe 3 O 4 -PEG-GA in 4.85 µg/mL was calculated to be 1.697 µg/mL based on 35% GA loading. The IC 50 values are given in Table 1.

Discussion
In this study we have redesigned our previously reported an anticancer nanocomposite formulation of Gallic acid loaded on iron oxide magnetite nanoparticles coated with polyethylene glycol ( [31]. The Percentage GA loading is found to be 35% in this redesigned nanocomposite is much higher compared to our previously reported nanocomposite of (Fe 3 O 4 -PEG-GA) with 7%. In addition to this in vitro release of GA from the nanocomposite was found to be highly sustained which took about 200 h (about 8 days) in human body simulated phosphate buffer saline (PBS) solution of pH 7.4 (blood pH) and pH 4.8 (intracellular lysosomal pH) at human body temperature 37 • C. In this study we tested the free drug GA, empty nanocarrier and the anticancer nanocomposite formulation Fe 3 O 4 -PEG-GA against A549 human lung carcinoma cells, HT29 human colon adenocarcinoma cell line, repeated on MCF-7 breast cancer cells and with normal 3T3 cells for incubation period of 24, 48 and 72 h. The IC 50 of the designed nanocomposite Fe 3 O 4 -PEG-GA are found to be much lower compared to free drug GA. The effective IC 50 which is based on percentage drug (GA) loading in nanocomposite Fe 3 O 4 -PEG-GA which is even further lower. Table 1 shows the details of the IC 50 against A549 human lung carcinoma cells, HT29 human colon adenocarcinoma cell line, and MCF-7 breast cancer cells and with normal 3T3 cells.

Chemicals
Gallic acid of 97% purity, iron oxide coated with polyethylene glycol (PEG) nanocarrier (Fe 3 O 4 -PEG) and gallic acid-iron oxide coated with PEG nanocomposite (Fe 3 O 4 -PEG-GA) were provided by the Material Synthesis and Characterization Laboratory, Institute of Advance Technology University Putra Malaysia (Serdang Selangor Malaysia). All three drugs were used for the preliminary screening of the effectiveness between gallic acid nanocomposite and pure gallic acid against normal cell and three different types of cancer cell lines. To prepare the stock solution, 5 mg of each drug was initially dissolved in 200 µL of dimethyl sulfoxide (DMSO) before the mixture was vortexed and sonicated for at least 30 min to ensure that the drug was completely dissolved. Upon sonication, 800 µL of RPMI 1640 (Nacalai Tesque, Kyoto, Japan) was added and then vortexed for 2 min to make the total volume of 1 mL. The drug sub stock was further diluted to a series of concentrations (0.47-200 µg/mL) and was used on the same day it was prepared. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and Trypsin-EDTA (Ethylene diamine tetraacetate) (0.25%) were purchased from Nacalai Tesque (Kyoto, Japan).

Cell Lines
Normal human fibroblast cells (3T3), human lung cancer epithelial cells (A549), human breast cancer epithelial cells (MCF-7) and human colon cancer epithelial cells (HT-29) were obtained from the American Tissue Culture Collection (Manassas, VA, USA). The cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Nacalai Tesque, Kyoto, Japan). The growth medium was supplemented with 10% fetal bovine serum, L-glutamine 15 mmol/L, penicillin 100 U/mL and streptomycin 100 µg/mL. All cells were incubated at 37 • C in humidified 5% CO 2 /95% air and the media was replaced every 2 to 3 days.

In Vitro Cell Viability Assay
The tetrazolium-based Colorimetric Assay (MTT) was used to determine the cytotoxicity of the drugs on the cancer and normal cell lines. In the case of viable cells in the in vitro MTT assay, the (nicotinamide adenine dinucleotide phosphate) NADPH-dependent oxidoreductase enzyme that is produced by the healthy mitochondria of a living cell will be secreted outside the cell membrane and converts the tetrazolium dye into a purple colored compound called formazan. Cells were first maintained in drug free media until the cells reached 80-90% confluency. MTT assay was initiated by the seeding process of plating 1 × Iron oxide magnetite nanoparticles (Fe 3 O 4 ) were prepared by previously reported method, in brief in 80 mL deionized water containing 6 mL ammonia hydroxide (25%) a mixture of 2.43 g (FeCl 2 ·4H 2 O) and 0.99 g (FeCl 3 ·6H 2 O) was added. After adding the above material sample was subject to ultrasonication for 1 h. Sample was centrifuged and washed thoroughly with water and dried in oven at 80 • C for 24 h and ground to fine powder [15,26,31]. PEG coating was carried out by dissolving the 0.2 g of dried iron oxide magnetite nanoparticles (Fe 3 O 4 ) in 1% of PEG solution followed by stirring for one hour after that sample was centrifuged and washed with ethanol thoroughly. GA was loaded on the designed nanocarrier Fe 3 O 4 -PEG by putting this sample to 50 mL (40 mL methanol and 10 mL water) of 1% GA and stirred for 24 h resulting in the formation of Fe 3 O 4 -PEG-GA. Next day sample was washed thoroughly with methanol and water by centrifugation. After that sample was dried in oven and ground to powder and subjected to further characterization.

Conclusions
The designed magnetite nanocomposite Fe 3 O 4 -PEG-GA was found to have higher drug GA loading 35% compared to previously reported nanocomposite with 7% GA loading. The in vitro release in human physiological pH 7.4 and pH 4.8 at 37 • C was conducted and was found to be highly sustained for up to 8 days compared to previously reported nanocomposite which took 3.5 days for the complete release and that was conducted at room temperature. In this study the magnetite nanocomposite Fe 3 O 4 -PEG-GA was found to be biocompatible with normal 3T3 cells. Both GA and Fe 3 O 4 -PEG-GA nanocomposite displayed time and dose-dependent anticancer activity against A549, MCF-7 and HT-29 cells Most importantly the IC 50 of the magnetite nanocomposite Fe 3 O 4 -PEG-GA were found against these cell lines were much better than free drug GA. These in vitro study results are highly encouraging to go further animal studies.