Synthesis, Characterization, and Cytotoxicity Evaluation of Gallic Acid Nanoparticles Towards Breast T47D Cancer Cells

3,4,5-trihydroxybenzoic acid, also known as gallic acid (Figure 1), is a polyphenolic compound found in plants and fruits such as mangoes, grapeseeds, raspberry, etc. Gallic acid is known to have anticancer, antimicrobial, and antiviral properties.14A research by Wang et al. in 2014 showed that gallic acid has anticancer effects towards MCF7 breast cancer cells by inhibiting the cancer cells proliferation and inducing apoptosis. Gallic acid works by activating the Fas/FasL apoptotic pathway. In addition, gallic acid also induces apoptosis through mitochondrial pathway. Gallic acid is hydrophilic, causing it difficult to penetrate into the wall of cancer cells.5 Preparation of gallic acid in form of nanoparticles is believed to increase the hydrophobicity. Therefore, this study aimed to synthesize nanoparticle of gallic acid coating with alginate-chitosan (Figure 1), and evaluate its cytotoxicity toward breast T47D cancer cells. Gallic acid coated alginate-chitosan nanoparticle is expected to be able to diffuse easily through the cancer cell membrane, that may lead to the increasing in its absorption and bioavailability, as well as the improvement of its anticancer activity.


INTRODUCTION
3,4,5-trihydroxybenzoic acid, also known as gallic acid (Figure 1), is a polyphenolic compound found in plants and fruits such as mangoes, grapeseeds, raspberry, etc. Gallic acid is known to have anticancer, antimicrobial, and antiviral properties. [1][2][3][4] A research by Wang et al. in 2014 showed that gallic acid has anticancer effects towards MCF-7 breast cancer cells by inhibiting the cancer cells proliferation and inducing apoptosis. Gallic acid works by activating the Fas/FasL apoptotic pathway. In addition, gallic acid also induces apoptosis through mitochondrial pathway. Gallic acid is hydrophilic, causing it difficult to penetrate into the wall of cancer cells. 5 Preparation of gallic acid in form of nanoparticles is believed to increase the hydrophobicity. Therefore, this study aimed to synthesize nanoparticle of gallic acid coating with alginate-chitosan (Figure 1), and evaluate its cytotoxicity toward breast T47D cancer cells. Gallic acid coated alginate-chitosan nanoparticle is expected to be able to diffuse easily through the cancer cell membrane, that may lead to the increasing in its absorption and bioavailability, as well as the improvement of its anticancer activity.
Nanoparticles are particles between 1-1000 nanometre in size. In the field of Pharmacy, nanoparticles have two meanings/interpretations, namely the nanometre-sized drug compounds or nanocrystal, and drug compounds that are encapsulated in nanometre-sized carrier system termed as nanocarriers. 6 There are several types of nanocarriers, such as nanotubes, liposomes, solid lipid nanoparticles, polymeric nanoparticles, etc. 7 The carrier polymer can be chitosan, which is a polysaccharide derived from chitin deacetylation process. Chitosan can be utilized in mucoadhesive drug delivery systems because its positively charged polymer chains can interact electrostatically with the negatively charged mucose. 8 In 2018, Adhikari and Yadav reported that chitosan and its derivatives had anticancer activity through cellular enzymatic, antiangiogenic and apoptotic pathways. 9 Whereas alginate, is a polysacharride polymer that can be obtained from brown algae with α-L-guluronic acid and β-D-mannuronic acid as the constituent monomers. Alginate is easily dissolved and degraded under normal physiological conditions, making it suitable to be used as systemic drug delivery. 10 Chitosan-alginate nanoparticles can be prepared, for example, by using the ionic gelation method. This method has some advantages such as simple preparation process with mild conditions without using hazardous or toxic organic solvents and without using high temperature heating process that may cause the decomposition of active compounds, so that this method is suitable to be used for the preparation of nanoparticles that contain thermolabile active compounds. 11 Nanoparticles have several advantages and disadvantages. The advantages of nanoparticles include: the capability to overcome physiological barrier in the body that is caused by the drug delivery system which is influenced by the particle size; increase the solubility of compounds that are poorly water-soluble so that it increases the bioavailability, active compounds stabilility, efficiency of drug distribution and allows better penetration in tumors with pores ranging 100-1000 nm in diameter. However, the disadvantages of nanoparticles are that they are easily aggregated so that it is difficult in handling and storage; small in size and not suitable for drugs that require large dosage; due to the nanometre size, able to penetrate undesirable parts such as nuclear envelope and cause genetic damage or mutations. 8 Nanoparticles characterization to determine the physicochemical properties, size, shape, and particles distribution can be done by using some instruments, namely Transmission Electron Research about synthesis of gallic acid and its derivatives nanoparticles is still very limited. In 2010, Moreno-Alvarez et al. reported that preparation of gold-gallic acid nanoparticles has antibacterial activity. 13 Li and Niu (2015) successfully synthesized silver-gallic acid nanoparticles that have high antimicrobial and low cytotoxicity activities towards normal cells. 14 Radoman et al. (2015) reported the synthesis and characterization of TiO 2 nanoparticles modified with octyl gallate 15 , whilst the latest research by Cordova et al. in 2017 showed that solid lipid-octyl gallate nanoparticles can improve the antimetastatic activity in mice model of lung cancer. 16 Synthesis and characterization of gallic acid nanoparticles with ionic gelation methods, as well as study to determine the its invitro anticancer activity has never been reported. This serves as the novelty aspect of this research.

Chemicals
Gallic acid, alginate, chitosan, and doxorubicin were purchased from Sigma-Aldrich Chemical Company.

METHODS
Synthesis procedure of gallic acid nanoparticles with ionic gelation method 100 mg of CaCl 2 is dissolved in 50 mL distilled water, then 50 mg of gallic acid is added, stir it until the solution is homogenized (Solution 1). Amount of 200 mg of Sodium alginate is dissolved in 25 mL distilled water. The pH is adjusted until 5.1 with 0.01 M HCl (Solution 2). Subsequently, the solution 1 is dropped into Solution 2 by using a syringe. Then, stir at the speed of 1400 rpm for 24 hours (Solution A). Amount of 100 mg of chitosan is dissolved in 25 mL of 1% (v/v) of glacial acetic acid. The pH is adjusted until 5.5 with 1N NaOH, then added 0.31 g of Tween 80, and stir it for 24 hours at 60 o C (Solution B). Solution B is then dropped into Solution A by using syringe whilst being stirred at the speed of 1300 rpm for an hour. The mixture is then centrifuged at 3000 rpm for 10 minutes until the nanoparticles are obtained in the form of pellets. The synthesized nanoparticle were freeze dried prior to use for analysis. Alginate-chitosan nanoparticles were prepared by using the same procedure with the synthesis of gallic acid nanoparticles, but without the addition of gallic acid.

Procedure of in vitro cytotoxicity determation of the nanoparticles by MTT assay
Breast T47D cells are seeded in RPMI 1640 (Gibco, USA) culture medium, which has been supplemented with 10% fetal bovine serum (Gibco, USA). Then, it is incubated at 37 o C in a humidified atmosphere of 4% CO 2 . The cell viability is determined by 0.1% trypan blue method. The test sample (nanoparticle) is diluted to reach the final concentration are 51.2; 25.6; 12.8; 6.4; 3.2; 1.6; 0.8; 0.4 μg/mL. Diluted samples were added to the target cells, and incubated for 48 hours. Amount of 100 μl of 5 mg/mL of MTT phosphate-buffered saline (PBS) was then added into the target cells of breast T47D in well plate, and the mixtures were reincubated for 4 hours. The mixtureswere then centrifuged, the medium is separated. DMSO in amount of 200 μl is added to each well to dissolve the blue purple-colored sediments. The absorbance is measured at 590 nm on a microplate reader model 550 (Bio-Rad, USA). The inhibition (in %) was calculated by using the formula below: Cytotoxicities of the nanoparticles are expressed by median inhibitory concentration (IC 50 ) value. The results will be compared with free-gallic acid (gallic acid not in form of nanoparticle) and doxorubicin as a positive control.

RESULTS AND DISCUSSION
In this work, chitosan polymer is used as nanocarrier. Chitosan is nontoxic, biocompatible, and biodegradable, but it is very fragile. Therefore, it requires alginate as a cross-linker to make it more stable. It has been reported that biopolymeric alginate-chitosan nanoparticle is effective and stable as an anticancer drug delivery 17,18 , in which, inspired us to perform the synthesis of gallic acid nanoparticles coating with alginatechitosan biopolymer.
Analysis of % yield of the synthesized gallic acid nanoparticles by UV-vis spectrophotometry Liquid (filtrate) obtained from centrifugation of the mixture of the synthesized nanoparticles was analyzed by UV-Vis spectroscopy at 690 nm to determine the concentration of free-gallic acid (gallic acid which did not convert into nanoparticles). Absorbance data of gallic acid standar solution are displayed in Table 1. Calibration curve of linear regression with the equation: y = 0.005x -0.0062, is obtained by plotting absorbance (mean value from three replication) of gallic acid in Y axis with the concentration of gallic acid (ppm) in X axis (Figure 2). The concentration of free-gallic acid (x = 20.4867 ppm) is generated by substituting Y in linear line equation of Y=0,005x-0.0062 with mean absorbance of filtrate (0.0962). The initial concentration of gallic acid is 500 ppm, thus concentration of gallic acid converted into nanoparticle is 479.5133 ppm (500 ppm-20.4867 ppm). So that, % yield of synthesized gallic acid nanoparticle is (479.5133/500)x100%=96%.

TEM analysis of gallic acid nanoparticles
TEM (Transmission electron microscopy) and SEM (Scanning electron microscopy) can help to identify the shape and size of small particles and nanoparticles. Compared to SEM, TEM has higher resolution. TEM could resolve object near the atomic level, as close as 1 nm. Besides that, SEM's magnifying power is up to 50,000 times, whereas TEM's magnifying power is up to 2 million times. 19 Therefore, in this work, identification of morphology and size of the synthesized nanoparticle using TEM.
TEM analysis of the nanoparticles (Figure 3) were conducted in Laboratory of TEM and Histology, Ejkman Institute, Jakarta, Indonesia, by using JEOL TEM 1010, 80 KV, with magnification of 40,000x. As shown, gallic acid nanoparticles (Figure 3a) have a spherical morphology with the size of 100-200 nm. Whereas alginate-chitosan nanoparticles (Figure 3b) also have a spherical shape with slightly smaller nanosize.

FTIR analysis of gallic acid nanoparticles
FTIR is used to confirm the molecular interaction between gallic acid and chitosan-alginate nanocarrier. FTIR analysis of the nanoparticles are displayed in Figure 4a-c. FTIR spectrum of gallic acid (4a) showed sharp band of hydroxyl (-OH) stretching aromatic at 3200-3500 cm -1 , absorption of carbonyl (-C=O) group at 1702 cm -1 , and absorption of aromatic carbon at around 1500-1600 cm -1 . Whereas, FTIR spectrum of alginate-chitosan nanoparticle (4b) showed N-H stretching vibration of amine group overlapped with carbonyl (-C=O) group of alginate at around 1500-1600 cm -1 , as well as a broad hydroxyl (-OH) stretching and amine group (-N-H) band at 3200-3600 cm -1 , which indicated the electrostatic interaction and hydrogen bonding between alginate and chitosan. Moreover, a very broad absorption band at 3000-3700 cm -1 in FTIR spectrum of gallic acid nanoparticle (4c) has confirmed the presence of hydrogen bonding between gallic acid and chitosanalginate nanocarrier.

Cytotoxicity of the nanoparticles towards breast T47D cancer cells
Studies on synthesis of gallic acid nanoparticle are limited. Daduang et al. in 2015 reported the synthesis of gold nanoparticles conjugated with gallic acid (GNPs-GA), and found that GNPs-GA inhibited cervical cancer cells less effective than gallic acid, but it was not toxic against normal vero cells, which indicated that GNPs-GA could be an alternative for cervical cancer treatment with less side effects to the normal cell. 20 Another researcher, Hu et al. (2015) reported that nanoparticles of gallic   Related to previous works, in this current research we examined in vitro anticancer activity of gallic acid coating with alginatechitosan nanoparticles against breast T47D cells. Table 2 summarizes cytotoxicities of gallic acid coating with alginate-chitosan nanoparticle, alginate-chitosan nanoparticle, gallic acid compound (not in form of nanoparticle), and doxorubicin. As displayed in Table 2, doxorubicin as a positive control has the lowest IC 50 value, exhibited the strongest cytotoxicity on T47D cells. Compared to gallic acid (IC 50 : 20.86 μg/mL) and alginate-chitosan nanoparticle (IC 50 : 38.46 μg/mL), gallic acid coating with alginate-chitosan nanoparticles exhibited greater cytotoxicity on breast T47D cells (IC 50 : 9.03 μg/mL). This result suggesting that compared to gallic acid compound, gallic acid nanoparticles coating with alginate-chitosan was successfully improved its cytotoxicity against breast T47D cells, due to the increasing in its hydrophobicity. Thus, it should be further developed as a promising candidate for treatment of breast cancer.

CONCLUSION
Gallic acid nanoparticle coating with alginate-chitosan has been successfully synthesized with ionic gelation method in 96% of yield. Gallic acid nanoparticle exhibited a strong cytotoxicity towards breast T47D cancer cells with IC 50 value of 9.03 µg/mL, which is potential to be developed as a candidate for new anti-breast cancer agent.  *IC 50 is the 50% half maximal inhibitory activity in µg/mL,expressed in mean value (n=3) +SD (standard deviation)