Biogenic Synthesis of Silver Chloride Nanoparticles from Padina gymnospora and their Evaluation of Antimicrobial, Larvicidal, Hemolytic and Anticancer Activities.

In the current study, silver chloride nanoparticles (AgClNPs) have been synthesized using the aqueous extract of Padina gymnospora and further characterized by Uv-visible spectroscopy, Fourier-transform infrared spectroscopy, X-ray powder diffraction, Scanning electron microscopy, energy dispersive spectroscopy, Transmission electron microscopy and Atomic force microscopy. The synthesized silver chloride nanoparticles were found to be mono-dispersed and spherical with an average size between 11.5 to 32.86 nm. The particles also showed a cytotoxic effect in a dose-dependent manner against MCF 7 cells (IC 50 = 31.37 µg/mL). Besides, it showed the larvicidal activity against Aedes aegypti at a lower dose (3.92 µg/ml) than that of the aqueous extract (13.01 µg/ml). It also exhibited significant antimicrobial activity against selected bacterial and fungal pathogens. The synthesized silver chloride nanoparticles showed the best minimum inhibitory concentration (MIC) of 8 mg/L in Canadian Albicans , followed by vancomycin resistance Enterococcus feacasli (VREF) 32 mg/L. Furthermore, the toxicity assessment by haemolytic assay revealed that AgClNPs could be safe and also used for further investigation. Therefore, the synthesized silver chloride nanoparticles may be used as a therapeutic agent for breast cancer, antimicrobial agent and also to control the dengue vector as attributed to the presence of bioactive components.


Introduction
Nanotechnology plays a diverse role in medical sciences and pharmaceutical industries in recent years [1]. In connection with that, evolving nano-based therapeutics has received substantial attention over the last two decades [2][3][4][5]. Researchers are tried to design the synthesis of nanoparticles for various applications which include therapeutics and diagnostics purposes, based on their cellular mechanisms and the advanced technology in nanomaterials [1]. Besides, silver-based nanoparticles have received more attention, due to their enhanced properties of physio-chemical and biological characteristics based upon the preparation of the nanoparticles by using additional elements [6]. Silver ions can inhibit bacterial multiplication by binding and denaturing bacterial DNA, thus affecting the ribosomal subunit protein and some enzymes inevitable for bacterial cell growth by penetrating the cells [7,8]. Most of the silver nanoparticles are used as anticancer [9][10][11], antimicrobial agents [6,12,13] and also have been reported for many biomedical applications [14]. The fusion of silver nanoparticles with natural resources is observed as an eco-friendly and cost-effective approach that bounces progressively over the other physico-chemical methods.
In the middle of associated silver materials, silver chloride plays an essential role in the application of wound healing, antimicrobial and also acts as a preservative of drinking water for long term storage in the tank [15][16][17]. Silver chloride nanoparticles (AgClNPs) also have various fields of photochemical and biomedical applications such as fabricating antiseptic catheters, bone cement, and fabrics owing to the antibacterial activity [18]. Despite these advantages, AgClNPs preparation is limited in a few ways such as micro-emulsion technique, ultrasound irradiation, matrix-based technique or mixing silver nitrate (AgNO 3 ) with hydrochloride acid in the presence of poly (vinyl pyrrolidone) for tuning its properties and applications [6].
Several protocols have been reported for AgClNPs synthesis using microorganisms such as bacteria, fungi and algae [6] [19]. To this extent, very few reports were available under the criteria of the synthesis of AgClNPs from marine resources and their application in the medicinal field [20]. The present work involves the synthesis of AgClNPs using the marine algae, P. gymnospora and its applications in the biomedical field have been investigated.
Padina gymnospora is a marine algae which belong to the family Dictyotacaea. It has a morphology of brown and darker at the base and the fan-shaped lobe has split into wedge-shaped pieces. It is used as a prominent natural wound-care product [21]. In the present study, it was initiated to explore the application of green synthesized AgClNPs by using seaweed P. gymnospora (Pg-AgClNPs) in the medical application of anticancer, antibacterial and larvicidal activity. In addition to that, the toxicity effect of Pg-AgClNPs was assessed.

Plant Materials and Chemicals
Seaweeds (Padina gymnospora) collected from Gulf of Mannar region, Tuticorin located (8.76°42'' N; 78.13° 48'' E) at the Southeast coast of India by handpicking and rinsing immediately with water to take away all kinds of epiphytes and other impurities (i.e., sand, molluscs, seagrasses, etc.). Then they were packed into sterilized Ziploc bags and transferred to the laboratory. Silver nitrate, cell culture medium, trypsin, and FBS were procured from HiMedia, Mumbai, India. Other chemicals/reagents used in this study were purchased from HiMedia, Mumbai, India.

Preparation of seaweed extract
The collected algae were cleaned with de-ionized water and allowed to shade dry up to seven days, and they were made into powder form using a blender. For the seaweed extraction, ten grams of algal powder was added into every three Erlenmeyer flasks containing 100 ml of double distilled water and mixed well and kept for 5 hrs without disturbance. Then they filtered through Whatman No.1 filter paper. The filtrates were used for the synthesis of AgNP for larvicidal activity against Ae. aegypti.

Synthesis of AgNP from seaweed extract
For AgNP synthesis, silver nitrate is used as a precursor. The AgNO 3 and extracts of P. gymnospora were selected for the production of green synthesis nanoparticles. Previously, AgNO 3 (1 mM) dissolved in distilled water (100 ml). The extract of P. gymnospora was added to AgNO 3 solution in 2:10 ratio, and the solution was incubated in a dark room and used for analysis. After reduction, the incubated solution centrifuged at 10,000 rpm for 15 min at 4 °C and three repeated wash with double distilled water was performed to discard a clear supernatant solution.

UV-Vis Spectra
The colour change was observed in the silver nitrate solution incubated with aqueous extract of P. gymnospora. The bioreduction of AgNO 3 ions in solution was monitored by periodic sampling of aliquots (0.1 mL) of aqueous components and measuring the UV-vis spectra of the solution in 10-mm-optical-path-length quartz cuvettes with an UV-1601 (Shimadzu, International, Co. Ltd, Tokyo, Japan) spectrophotometer at a resolution of 1 nm between 400 and 800 nm with a scanning speed of 1,856 nm/min.

XRD analysis
The XRD analysis was carried out to analyze the crystallite size of Pg-AgClNPs by the biological method. Samples for powder X-ray Diffraction (XRD) were prepared by making a thin film of powder with ethanol on a glass plate, and the measurement was performed using an Ultima IV -Rigaku diffractometer with CuKα radiation (λ = 1.540 Å, 45 kV and 30 mA) Tokyo, Japan.

FT-IR analysis
FTIR analysis was performed using an FTIR-8400S spectrometer (Shimadzu, International, Co. Ltd, Tokyo, Japan) spectrophotometer to verify possible interaction of chemical bonds between bio-molecules of seaweed and silver nitrate solution. Samples were scanned from 400-4000 cm − 1 with potassium bromide pellets. Characteristic peaks of plain Pg-AgClNPs, seaweed extract and are expressed in a reciprocal wavelength (cm − 1 ).

TEM analysis
Conventional TEM analysis was performed in a High-Resolution Transmission Electron Microscope (HR-TEM -JEOL-2100 + JEOL India Pvt. Ltd., New Delhi. India) using a magnification of 46000×. The samples were prepared by the formvar resin grid method. Briefly, a 0.5% w/v suspension of prepared Pg-AgClNPs was sprayed onto a formvar resin-coated TEM grid and air-dried for 10 minutes before observation. The morphology of Pg-AgClNPs complexes was photographed.

SEM and EDX analysis
For morphological characteristics, the Pg-AgClNPs were uniformly spread and sputter-coated with platinum using an ion coater for 120 s, and then observed under an SEM EVO18-CARL ZEISS, USA. EDX is a technique used for identifying the elemental composition of the specimen or an area of interest thereof, and the system works as an integrated feature of the SEM which was done by using Quantax 200 with X-Flash® 6130. Bruker India Scientific Pvt. Ltd. Bangalore, India.

AFM analysis
A thin film of the sample was prepared on a coverslip by dropping 0.1 ml of the sample on the slide and allowed to dry for 30 min. The slides were then scanned with AFM (Park system XE-100 AFM system, Suwon, Korea). The AFM characterization was carried out in ambient temperature in non-contact mode using silicon nitrate tips with varying resonance frequencies.

MTT-based cytotoxicity assay
The cytotoxic effects of compounds against human tumour cell lines were determined by a rapid colourimetric assay, using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) and compared with untreated controls. For screening experiments, the cells were seeded in 96-well plates in 100 µL of medium containing 5% FBS, at plating density 10,000 cells/well and incubated at 37 °C, 5% CO 2 , 95% air and relative humidity (100%) for 48 h prior to addition of compounds. After 48 h, compounds at various concentrations were added and incubated at 37 °C, 5% CO 2 , 95% air and relative humidity (100%) at 48 h. Triplicate was maintained, and the medium without the sample was served as control [22].

Assessment of cell death through Flow cytometry
Flow cytometry study was carried out using propidium iodide staining. Breast cancer cell line MCF 7 was seeded in 6 wells with microtiter plates. After 24 h, the medium was discarded, and the calculated concentration of the compound was added to each well and incubated for 48 h at 37 °C. Cells were washed with PBS several times and trypsinized. The cells were then suspended in 1 mL of PBS and centrifuged at 1000 rpm for 3 min. The supernatant was discarded, and the pellet obtained was vortexed at a lower speed. Then, 1 mL of PBS was added again and centrifuged at 1000 rpm for 6 min. The supernatant was discarded, and the pellet was suspended in 250 µL of PBS. The obtained cell suspension was stored in 0.8 mL of ethanol and incubated at 4 °C for 30 min. To the sample, 10 µL of propidium iodide was added and incubated at room temperature for 30 min and protected from light (BD, Sasc, Jazz Trademark, US).

Mosquito rearing
The egg rafts of Ae. aegypti obtained from the Vector Control Research Centre, Madurai, Tamil Nadu India. Larvae of A. aegypti reared in enamel trays containing dechlorinated water. The larvae are fed with a finely powdered mixture having a 3:1 ratio of dark biscuits and dry yeast. The larvae were observed and monitored for III instar stages, and it was used for the larvicidal activity.

Larvicidal activity
The toxic activity of different concentrations (2, 4, 6. 8 and 10 µg/ml) of Pg-AgClNPs was evaluated against the III instar of A. aegypti. The aqueous extract of P. gymnospora was tested against various concentrations (5,10,20,30, and 60 µg/ml) against the larvae. Mortality recorded after 24 and 48 hrs of post-exposure [23]. The results obtained were subjected to statistical analysis. The dose-dependent response data were concerned with probit analysis for finding the LC 50, upper, and lower confidence limit at 95% [24].

Anti-microbial activity
The antimicrobial activity of Pg-AgClNPs was done by the well diffusion (or) Kirby-Bauer method in Muller-Hinton Agar (MHA) medium. The sterilized MHA medium at 121 °C at 15 lbs for 20 min was poured into sterile Petri plates, allowed to solidify, and the pathogens were swabbed using a sterile cotton swab over the surface of the medium. After 15 min the wells were made using cork-borer on agar medium. The Pg-AgClNPs were loaded at three different concentrations dissolved in 50% Dimethyl sulfoxide (DMSO) 1 mg/mL, 2 mg/mL and 3 mg/mL as stock solution 100 µl each concentration was loaded at three different wells. Zone of inhibition was measured after 24 hrs of incubation at 37 °C.

Minimum Inhibitory Concentration (MIC) of synthesized nanoparticles
The experiments were carried out in 96 well plates (TARSONS, Kolkata, India). The flat bottom, 100µL of sterilized Muller-Hinton Broth was loaded in all the test wells. The synthesized nanoparticles and commercially available antibiotics were diluted 512, 256, 128, 64, 32, 16, 8, 4, 2, 1 mg/L. For positive control taken in ten wells, the 11th well for negative control (without organism) and 12th well for test pathogens as 5µL, The 12 hrs grown in MHB broth at concentrations of 10 5 to 10 6 CFU/mL were added to all the wells except negative control. The plates were incubated at 37 °C for 16 hrs. After incubation 10 µL of 5 mg/mL concentration, freshly prepared MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) was added to all the wells and incubated for 10 min. 100 µL of DMSO solution was added as the solubilizing agent and incubated for 15 minutes. Optical density (OD) was taken at 595 nm, the percentage of cell death was calculated, and MIC was noted.

Hemolytic assay
The hemolytic activity of Pg-AgClNPs was determined in fresh anti-coagulated human blood cells. The cells were washed twice with phosphate-buffered saline (PBS) and diluted at 10% of concentration, 800 µL of nanoparticles diluted at the concentrations of 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78 µg/mL in phosphate-buffered saline solution at micro-centrifuge tubes. Negative control as PBS and positive control 1% Triton X-100, 200 µL of Red Blood Cell (RBC) suspension added all tubes incubated for 1 hrs at 37 °C. All treated tubes centrifuged at 2000 rpm for 5 min and supernatants were transferred to the new 96-well microtiter test plate, and absorbance read at 540 nm. The amount of haemoglobin released was calculated using negative control (no hemolysis) and Triton X-100 (positive control, complete hemolysis).

Statistical Analysis
One-way analysis of variance (ANOVA) followed by a Tukey post-hoc test was used for MTT analysis. Probit analysis followed by Finney method. These statistical analyses were performed using the Statistical Package for the Social Sciences 16.0 package in Chicago, USA. Data were presented as mean ± SD and P values < 0.05 was considered to be statistically significant.

Result And Discussion
Initially, the formation of green synthesized Pg-AgClNPs was visually identified by the appearance of dark brown colour in 24 hrs of incubation (P. gymnospora with silver nitrate) (Fig. 1). The colour change from pale yellow to dark brown clearly indicating the formation of silver chloride nanoparticles. Then it was optically confirmed by UV-spectrophotometric analysis. A strong surface plasmon resonance peak was obtained around 377 nm (Fig. 2), which indicates the formation of AgClNPs [25,26]. The maximum absorbance indicates the relative size of the nanoparticle, where the higher number corresponds to a larger particle size [27]. The chloride ions present in the seaweed extract converts AgNO 3 to AgClNPs. Thus the chlorine content of the extracts could be the source for the formation of AgClNPs [28,29]. The chlorine ions in the seaweed extracts were confirmed by EDAX Analysis. It reveals the presence of silver (56.6%) and chloride (22.9%) along with other elements such as B, C, O, Na, and Si was found to be contributed by the bioorganic materials present in the seaweed extract bound to the nanoparticle surface (Fig. 3).
The FTIR spectrum of P. gymnospora (Fig. 4a) shows that the strong bands observed at 1100, 1137 and 1416 cm − 1 are assigned to -C-O, -C = O and -C = C stretching of aromatic acids and esters present in the seaweed extract respectively. The band at 1643 cm − 1 is attributed to -C = O stretching of amides. The sharp bands at 2307, 3399 and 3727 cm − 1 correspond to NH stretching vibrations of primary and secondary amines in the crude extract. In the fingerprint region, the peaks observed at 601 and 676 cm − 1 correspond to the presence of C-Cl groups in the seaweed extract. These functional groups could act as biomolecules for stabilizing, capping and reducing agents for the formation of AgClNPs. On the other hand, the IR spectrum of Pg-AgClNPs shows the peaks of -C-O stretching shifted from 1100 cm − 1 to 1088 cm − 1 and the peak of N-H stretching shifted from 3399 cm − 1 to 3142 cm − 1 (Fig. 4b). It reveals that the carboxylic and amine groups are involved in the stabilization and immobilization of AgClNPs in the seaweed extract. The role of -C = C in the reduction of silver salt is evident by the peak observed at 1392 cm − 1 which was found at 1416 cm − 1 in seaweed extract respectively. Further, the presence of peaks at 1633 cm − 1 confirms the amide stretching group. The sharp peak observed at 594 cm − 1 confirms the presence of AgCl NPs stabilised by the seaweed P. gymnospora extract.
The powder XRD analysis of synthesized Pg-AgClNPs (Fig. 5) exhibits the crystalline structure with diffraction peaks obtained at 27.87°, 32.35°, 46.20°, 54.45°, 57.45° and 67.49° corresponds to the planes of (111), (200), (220), (311), (222) and (400) respectively which matches with the standard JCPDS File No. 85-1355. There is no other peak indicating that the high purity of prepared Pg-AgClNPs and the strong intensity of the peak shows a high crystallinity of the sample. The grain size was calculated as 30.39 nm using Scherrer's formula. The absence of additional reflection indicates that Pg-AgClNP's lattice was not affected by other molecules in seaweed extract. SEM micrographs showed that the synthesized nanoparticles were observed with fine spherical crystals (Fig. 6) and the morphological features obtained from TEM reveal that the Pg-AgClNPs are monodispersed spherical shape with the average particle size of around 33 nm (Fig. 7). AFM micrographs indicate that the formulated Pg-AgClNPs possess a spherical shape and the calculated sizes in the range of 15.5 to 21.38 nm (Fig. 8). It characterizes the interactions between the Pg-AgClNPs and the supported lipid bilayers in real-time. The particle size is varied from 10 to 28 nm.
The Pg-AgClNPs were evaluated for anticancer activity against breast cancer cell line (MCF-7 cell line), and the cytotoxicity to the normal (Vero) cell line was also studied. Pg-AgClNPs showed a cytotoxic effect on breast cancer cells in a dose-dependent manner. The IC 50 values of Pg-AgClNPs against breast cancer cells were found 31.37 µg/mL for MCF-7 and 421.46 µg/mL for Vero Cells (non-tumor cells) (Fig. 9). In vitro cell viability assay clearly revealed that Pg-AgClNPs were severely toxic to human breast cancer cells even more than AgNPs (data not shown). Interestingly, the synthesized Pg-AgClNPs have lower cytotoxicity effect on Vero non-tumour derived cell line. Growth inhibition related to cell cycle arrest and apoptosis was determined in flow cytometry using propidium iodide. MCF7 cell lines were treated with IC 50 concentration of compounds. MCF7 cells treated with IC 50 concentration of compounds increased the percentage of apoptosis in G0-G1 phase (5.16% when compared with control of 1.39%). An increase in the percentage of apoptotic cells was measured by flow cytometry, clearly clarifying an induction of programmed cell death in the cancer cells after treatment with AgClNPs. The Pg-AgClNPs induce cell death through the mitochondrial pathway. Most of the studies reported that AgNP's mediated cell death is independent of p53 [30] [12,13]. Previously, it was reported that the biosynthesis of AgClNPs induces the cytotoxicity and apoptosis effect on human breast cancer cells through the up-regulation of apoptotic factors [6].
The larval bioassays performed by aqueous extracts of P. gymnospora and Pg-AgClNPs against larvae of Aedes agypti are a dengue vector. For the extract of P. gymnospora showed LC 50 of 34.92 µg/ml in 24 hrs and 21.61 µg/ml in 48 hrs and 11.82 µg/ml in 72 hrs. While the synthesized Pg-AgClNPs show LC 50 and LC 90 of 7.15 µg/ml in 24 hrs, 6.06 µg/ml, in 48 hrs and 3.61 µg/ml in 72 hrs (Table 1). Among them, the Pg-AgClNPs showed higher larvicidal activity against A. aegypti with low concentration compared to the aqueous extract of P. gymnospora. The toxicity mechanisms of mosquito mortality in the nanoparticle treatment were studied recently. It is hypothetically suggested that the mechanism of toxicity against mosquito larvae by the penetration of nanoparticles through the exoskeleton. In intracellular space, nanoparticle degrades the enzymes and organelles, and it leads to the loss of cellular function and cell death [31,32]. The AgNPs from Artemisia vulgaris leaf extracts showed the mosquito larvicidal activity. By the accumulation of nanoparticles in the midgut region of mosquito, larva causes damage in the midgut, cortex region, and epithelial cells. Similar to seaweed mediated synthesis of nanoparticles, several plants mediated synthesized nanoparticles were shown in mosquito larval control [33]. Table 1 The larval bioassays performed by aqueous extract of P. gymnospora and green synthesized silver chloride nanoparticle AgClNPs act as an alternative source for microbial pathogens. The green synthesized Pg-AgClNPs were assessed for the antimicrobial activity against the human pathogens. Pg-AgClNPs showed antimicrobial activity in a dosedependent manner. They were reported in Table 2. Compared to pathogenic bacteria and fungi, it acts as a good antifungal activity (Fig. 11). Moreover, the MIC also investigated these microbes by using Pg-AgClNPs at the range of 1 mg/mL by the serial dilution method. They were reported in Table 3. Among all concentrations of the selected pathogens, Canadian Albicans showed the best minimum inhibition at the concentrations of 8 mg/L, followed by VREF 32 mg/L. AgNPs act as an alternative source for microbial pathogens. Due to different variations and great impact on antimicrobial properties, it was predominately used in pharmaceuticals and drug industries [34]. Pg-AgClNPs showed antimicrobial activity in a dose-dependent manner. Compared to pathogenic bacteria and fungi, it acts as a good antifungal activity. Microbial growth inhibition in AgNPs is due to the surface and particle size variance [35]. The possible mechanism of antimicrobial activity is because of the generation of free radicals. The AgNPs interact with bacteria and release the Ag + ions inside the cell as leads to cells leaked out caused by protein denaturation [36,37]. The small size of AgNPs has a larger surface area, facilitates the interaction with the bacterial cell membrane, and altered the primary function of the cell membrane, including permeability and cell respiration, causing cell apoptosis [38]. Previous studies reported that the bacterial cells treated with AgClNPs caused the morphological changes and that AgClNPs were distributed on the surface of the bacterial cells [29].  Hemolytic activity of synthesized silver nanoparticles was screened at different concentrations among them. Pg-AgClNPs particles showed the hemolytic activity in 100 µg/mL 92% followed by 50 µg/mL 12% of hemolysis from the 25 µg/ml concentration showed no hemolysis to the human blood cells (Table 4). This result suggests that Pg-AgClNPs can be safe and also used for further investigation (Fig. 12). At the nanotoxicity level, the investigation on blood compatibility is essential because the blood cells are affected by nanoparticles directly or indirectly. The erythrocytes circulate to various organs through the cardiovascular system leading to DNA damage, cell membrane injury, and congenital malformation [7,8]. In this condition, the biocompatibility of AgNPs rupturing and releasing of erythrocytes have more attention to analyzing the toxicity level of nanoproducts [34].

Conclusion
The active principle of AgClNPs obtained from the seaweed P. gymnopora exhibits potent anticancer activity against MCF 7 cells, and it also has a larvicidal activity against the A. aegypti as compared to the aqueous extract of P. gymnopora without harming the non-target organism. AgClNPs also exhibits antimicrobial activity and biocompatibility to RBC in connection with human welfare. Overall, the synthesis of Pg-AgClNPs is considered as eco-toxic and eco-friendly. It can be effectively used against breast cancer cells. In addition, it controls the A. aegypti dengue causes vector disease, and it may also be used as an antimicrobial agent against the human infectious pathogen.

ACKNOWLEDGEMENT
We are grateful for the management and principal of the Ayya Nadar Janaki Ammal College, Sivakasi for providing good laboratory facilities.   TEM analysis of Pg-AgCLlNPs. TEM micrograph showed that the presence of mono-dispersed spherical shape of nanoparticles was the average size of 32.866nm.  The IC50 values of Pg-AgClNPs against breast cancer cells were found 31.37µg/mL for MCF-7 and 421.46µg/mL for Vero Cells (non-tumor cells).  Zone of inhibition of Pg-AgClNPs against human bacterial and fungal pathogens of clinical isolates.   Pg-AgClNPs action illustrating the possible mechanism of the delivery system of noncombinations induced by anti-cancer activity

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