Synthesis and Characterization of Mangiferin Loaded N,O-CMC Nanoparticles and its Cytotoxic Effect on Osteosarcoma MG-63 Cells

Mangiferin is a xanthone glycoside, naturally isolated from Mangifera indica . Mangiferin has been reported for a wide range of pharmacological activities and its anticancer potential is very well known. However, the mangiferin anti-cancer potency is inadequate due to its poor water solubility. N,O-Carboxymethyl Chitosan (N,O-CMC) is a smart biopolymer, in which its bio-compatible, biodegradable and non-toxic making it ideal for abundant biological applications include the delivery of lipid soluble drugs. Also use-ful to improve and replace biological tissues and gene therapy. Hence, this study attempts to synthesize and characterize mangiferin-N,O-CMC nanoparticles and evaluate its antioxidant and cytotoxic properties. The mangiferin-N,O-CMC nanoparticles were prepared by loading mangiferin into N,O-CMC nanoparticles and characterized by FT-IR, DLS, SEM, Zeta potential and XRD measurements. In-vitro antioxidant was carried out by the DPPH method. The cytotoxic effect of mangiferin-N,O-CMC nanoparticles was carried out on Osteosarcoma MG-63 and 3T3 cells by using MTT assay method. The synthesized mangiferin-N,O-CMC nanoparticles with particle size ranges from 200 (cid:6) 10 nm. The charge of N,O-CMC nanoparticles were con(cid:977)irmed by Zeta potential and found to be (cid:0) 45.8 mV. In the DPPH method, mangiferin-N,O-CMC nanoparticles showed IC 50 value between 7.8-15.6 (cid:22) g/ml. In MTT assay, mangiferin-N,O-CMC nanoparticles exhibited a signi(cid:977)icant reduction in the growth of osteosarcoma MG-63 cells and there is no toxic effect against normal 3T3 cells. These (cid:977)indings designated that the synthesized mangiferin-N,O-CMC nanoparticles were very ef(cid:977)icient nanocarrier in delivering the mangiferin to cancer cells.


INTRODUCTION
Mangiferin is a xanthone glycoside, mainly obtained from Mangifera indica and reported to possess many pharmacological effects that including antioxidant, analgesic, anti-in lammatory, antidiabetic, neuroprotective, hepatoprotective, cardioprotective and anticancer studies (Mahendran et al., 2014;Sekar, 2015). Over the past few decades, mangiferin has been comprehensively studied concerning the anticancer properties. There are few evidences strongly supported that mangiferin has been used to prevent the growth and development of cancer cells by modulation of many molecular pathways. The literature of mangiferin constantly showed that it has synergistic effects with chemotherapeutic agents include the etoposide, oxaliplatin and doxorubicin (Gold-Smith et al., 2016). Perhaps the most noticeable anti-proliferative effect of mangiferin on tumour cells has been detected in animal experimental models, where the tumour volume was reduced similarly with a standard drug cisplatin. Mangiferin also exhibits other promising features including low toxicity with wide oral safety margin, as pointed out by (Gold-Smith et al., 2016).
Nevertheless, mangiferin has low solubility in water which con ines its clinical ef icacy and bioavailability. (Hou et al., 2012), also reported that the absorption range of mangiferin was improved when large dose was administration. Also mentioned that the mangiferin pharmacokinetics pro ile was nonlinear in human, the major reason for poor bioavailability of this naturally isolated mangiferin is mainly because of its low water solubility and absorption. In this regard, (Othman and Sekar, 2019) attempted to increase its aqueous solubility and bioavailability by converting mangiferin into silver nanoparticles and studied for its in-vitro cytotoxicity study against cancer cells. However, the water solubility has not been achieved as expected when mangiferin was converted into silver nanoparticles.
Chitosan, a well-known amino polysaccharide with its biodegradability, biocompatibility and low cost. The interesting characteristics of chitosan have made it be widely used in pharmaceuticals, agriculture, medicine, food and biotechnology ields. Although chitosan is insoluble in water,its water solubility could be improved when it was converted into carboxymethyl chitosan (Yang et al., 2017). N,O-CMC has been prepared using chitosan, isopropanol, chloroacetic acid and sodium hydroxide by maintaining the temperature at 50-70 • C. It has abundant attractive physical and biological properties such as biocompatibility, moisture retention, aqueous solubility and gel-formation. All of these properties together make it that N,O-CMC as a promising biomaterial (De-Abreu and Campana-Filho, 2009). Hence, the present study aims to synthesize, characterize mangiferin loaded N,O-CMC nanoparticles and subsequently evaluating its effect against osteosarcoma MG63 cells. Along with that we also evaluated its antioxidant activity by DPPH method.

Isolation of Mangiferin from Mangifera indica
Mangiferin was isolated from Mangifera indica and puri ied based on our earlier published protocol (Othman and Sekar, 2019). The chemical structure of mangiferin is shown in Figure 1.

Preparation of N,O-CMC from Chitosan
2 g of Chitosan was mixed with 20 ml of isopropyl alcohol and prepared into the form of a slurry. This was followed by a dropwise addition of 10 ml of 5M NaOH solution. The reaction mixture was

Figure 4: Zeta Potential distribution of mangiferin loaded N,O-CMC nanoparticles
then stirred continuously for 3 h at 60 o C by adding monochloroacetic acid in dropwise slowly at consistent intermissions. After that, the solution was iltered and the residue was washed using enough quantity of methanol and dried in a hot air oven at 37 • C for 24 h. The dried sample was characterized and used for nanoparticle synthesis (Anitha et al., 2012).

In-vitro antioxidant activity by DPPH method
A 100 µl of mangiferin-N,O-CMC nanoparticle solution in different concentrations were added to 2 ml of 100 µM DPPH solution which was prepared earlier by using methanol. The reaction mixture was incubated at 37 o C for 20 min and the absorbance was determined at 490 nm using UV-visible spectrophotometer (Othman and Sekar, 2019). The percentage inhibition was calculated as follows,

In-vitro cytotoxic activity by MTT assay
The in-vitro cytotoxic study of mangiferinloaded N,O-CMC nanoparticles in four different concentrations (25, 50, 100 and 250 µg/ml) were carried out by MTT assay method using the standard procedure mentioned in our earlier published protocol (Othman and Sekar, 2019). The absorbance was measured using a microplate reader at 540 nm. Note that, control values were set at 100% viable and the respective concentrations were calculated and expressed as a percentage of the control.

Statistical Analysis
The values were expressed as mean±SD of three replicate measurements. The statistical analysis was carried out by one way ANOVA followed by multiple comparison test of Turkey-Kramer. P values <0.05 were considered as signi icant.

Characterization of Mangiferin Loaded N,O-CMC Nanoparticles
The size distribution of mangiferin-N,O-CMC nanoparticles obtained DLS . It is observed in Figure 2, that mangiferin-N,O-CMC nanoparticles lied within a size range of 200±10 nm. Based on the SEM investigations as shown in Figure 3, the morphology of mangiferin-N,O-CMC nanoparticles indicated spherical and lower type particles with a size range of 200 nm. Zeta potentials were measured for mangiferin-N,O-CMC nanoparticles and the value was found to be -45.8 mV (Figure 4).
The FT-IR spectrum of N,O-CMC, N,O-CMC nanoparticles, mangiferin and mangiferin-N,O-CMC nanoparticles were taken and compared in Figure 5. In the spectrum of N,O-CMC, the peak observed at 3265 cm −1 was due to -OH group, the peak at 1578 cm −1 was due to carboxylic group, and the peak at 1630 cm −1 was due to the presence of amino group. After developing nanoparticles, the peak at 1630 cm −1 was slightly shifted to 1636 cm −1 , con irming that the phosphate groups present in TPP undergoing a cross-linking reaction with the protonated amine groups of N,O-CMC (Anitha et al., 2012). As for mangiferin, the absorption band at 3362 cm −1 was due to the presence of -OH group, the sharp peak at 1648 cm −1 was assigned for C=O, the aromatic C=C peak was appeared at 1618 cm The XRD of mangiferin-N,O-CMC nanoparticles was taken to understand its physical nature and the result revealed that mangiferin-N,O-CMC nanoparticles did not comprise with any crystalline type of peaks. This might be probably due to the development of an amorphous complex during the formation of nanoparticle within the nanoparticle matrix as shown inFigure 6.
In the in-vitro antioxidant activity using the DPPH method, mangiferin-N,O-CMC nanoparticles showed potent antioxidant activity by increasing the concentrations (Table 1). The IC 50 value was found to be between 7.8-15.6 µg/ml. As shown in Figure 7, there was a colour change from purple to yellow in the DPPH solution, indicating that mangiferin-N,O-CMC nanoparticles having signi icant antioxidant activity at lower concentrations. This result was consistent with our previous study results of antioxidant activity of mangiferin in silver nanoparticles in the DPPH method.
In the present study, four different concentrations of mangiferin-N,O-CMC nanoparticles varying from 25 µg/ml to 250 µg/ml were tested for cytotoxicity study against Human MG-63 (osteosarcoma cells) by MTT assay (Figures 8, 9, 10 and 11). Mangiferin-N,O-CMC nanoparticles exhibiteda signi icant (P<0.001) reduction of cancerous cell growth in all the tested concentrations in a concentration dependent manner. Hence, for testing against normal cells (3T3), these concentrations were selected. There was no signi icant toxicity related with mangiferin-N,O-CMC nanoparticles with normal cells, thus indicating the safety of the synthesised nanoparticles.
Cancer is well recognized as a serious lifethreatening disease of old age. The conventionally used medicines for the treatment of cancer have some boundaries including non-speci ic targeting and biodistribution, poor bioavailability, lack of aqueous solubility and low therapeutic indices. Because of these reasons, those drugs cannot be used widely in clinical treatment. Mangiferin is a naturally isolated compound from Mangifera indica and well-known for its chemotherapeutic properties. However, it is yet been developed as a drug in different dosage forms due to its low bioavailability and lack of water solubility. To solve these problems, the application of nanotechnology in drug manufacturing has been considered as a promising strategy. In addition, the anticancer ef icacy of drug nanoparticles in-vivo and in-vitro toward various types of cancer has been reported in a plethora of studies (Wang et al., 2016).
For instance, the anticancer properties of O-CMCmetformin nanoparticles have been exhibited on pancreatic cancer cells (Snima et al., 2012). On the other hand, (Anitha et al., 2014) , also revealed that the synergistic anticancer activities of 5-luorouracil and curcumin-loaded N,O-CMC NPs towards colon cancer cells. In the present study, we successfully synthesized mangiferin-loaded N,O-CMC nanoparticles and characterized by DLS, SEM, FT-IR, Zeta potential and XRD measurements. In the DPPH method, mangiferin-loaded N,O-CMC nanoparticles showed signi icant antioxidant activity at lower concentrations. In MTT assay results indicated that the synthesized mangiferin-loaded N,O-CMC nanoparticles could inhibit the growth of Osteosarcoma MG-63 cells. These results were in the agreement with the previous study regarding the mangiferin silver nanoparticles in DPPH and MTT assay methods (Othman and Sekar, 2019).

CONCLUSIONS
In this study, mangiferin-N,O-CMC nanoparticles were prepared and characterized by FT-IR, DLS, SEM, Zeta potential and XRD. The results showed that mangiferin was effectively loaded into N,O-CMC nanoparticles with a size range of 200±10 nm. The synthesized mangiferin-N,O-CMC nanoparticles possessed potent antioxidant and cytotoxic properties in DPPH and MTT assay methods, respectively. These pilot study results demonstrated the ability of the mangiferin-N,O-CMC nanoparticles in carrying hydrophobic drugs, thus making it as an alternative promising candidate for drug-delivery applications. However, further studies are warranted to reduce the particle size of mangiferin-N,O-CMC nanoparticles and tested with other type of cancer cells to conirm its safety and ef icacy.