Synthesis of β-Glucan nanoparticles from red algae derived β-Glucan for potential biomedical applications

The present study highlighted that the synthesis of β-Glucan nanoparticles (β-GluNPs) developed as a facile method to prevent cancer and infectious diseases, which is highly effective and inexpensive in the biological eld. This research study has demonstrated the use of marine algae (Gracilaria corticate) to extract water-soluble β-Glucan and synthesis of β-GluNPs by dissolving the extracted β-Glucan in NaOH under optimal conditions. The molecular structure of the extracted and stabilized β-GluNPs was analysed using NMR. Further, the Physico-chemical parameters of β-GluNPs were analysed by the high throughput instruments like UV spectroscopy, FTIR, DLS, ZETA Potential, SEM, HRTEM, and XRD analysis. The synthesis of β-GluNPs conrmed by IR spectroscopy. The HR-TEM results demonstrated that the formation of polydispersed nanoparticles with a mean size of 20 ± 5 nm. The hydrostatic zeta potential was − 22.7mV, which indicated their colloidal stability. The XRD pattern revealed the crystalline nature of the nanoparticles. Besides, β-GluNPs performed better antibacterial activity against the tested pathogens. The apoptosis and DNA fragmentation observed to be IC 50 42.5 µg/ml of the β-GluNPs. The DNA fragmentation assay indicated the selective inhibition of the MCF-7 cell line by DNA damage. Hence, β-GluNPs used as a promising alternative drug against human breast cancer.


Introduction
Recently, the researchers were focusing on green synthesis and eco-friendly platform biomaterials [1]. The optimal green synthesized biomaterials are non-toxic and excellent biocompatibility. Nano biomedicine is quite a novel area that synthesis and applies nanosize biomaterials for biomedical applications. The conventional microbial producing polysaccharides are very effective due to their negative charge, which interacts with positively charged compounds [2]. Consequently, it allows different natural functionalities [3,4].
Marine algal based β Glucan i.e. polysaccharides made up of glucose present in red, green, and brown algae seaweeds are starch and cellulose, cellulose and laminarin and cellulose and oridean starch, respectively that are considered as superior bioactive molecules in biomedical science [5,6]. Besides, epidemiological and clinical trials alleged that marine algae β-glucan is an immunostimulant and a potential antitumor drug [7,8]. In recent years, the nanoparticles have been synthesized using polysaccharides such as chitosan and Glucan [9][10][11]. Glucans, a class of natural polysaccharides, are glycosidic bonds and differing degrees of branching from marine algae sources [12]. Biologically derived β-Glucans have potential antimicrobial properties against viral, bacterial, parasitic, and fungal pathogens [13]. Many researchers reported that the administration of natural β-Glucan could inhibit bacterial pathogens and increase cytokines, monocytes, and neutrophils [14,15].
Cancer is presently one of the main diseases responsible for human mortality and morbidity, which cause health and economic burdens. Cancer treatment is commonly based on chemotherapy and resistance to common chemotherapeutic agents and hazardous side effects highlight the importance of hunting for potent anticancer drugs. Hence, new drugs that improve cancer curability without any side effects on the host system are needed [16,17]. Plant derived drugs such as taxol and camptothecin have been used as anticancer drugs that bear high costs, and cannot be afforded by a common man [18]. Thus, new approaches and sources to nd alternative drugs that are effective and inexpensive are in focus.
Nanotechnology is rapidly emerging with its biological science application and recently, in medical science [19]. Nanoparticles (NPs) are explored in cancer biology (which leads to a separate discipline as nanooncology) to diagnose and treat human cancers [20,21]. The utilization of NPs will facilitate both tumor targeting and drug delivery in a distinct way [22]. Recent studies have con rmed that specially formulated metal NPs have shown anticancer activities [23,24]. The potential adverse effects of NPs on biological systems due to the unpredicted nature of metal NPs has raised questions about their applications in humans [25]. Therefore, the selection of nanomaterials for applications within the eld could also be a challenge as materials that are biodegradable, biocompatible, and non-toxic are desirable [26]. Nanoparticles synthesized from biopolymer bases possess advantages such as availability from replenishable marine algal resources, biodegradability, and biocompatibility [24]. Therefore, the present study was to biosynthesize water-soluble β-Glucan nanoparticles using Gracilaria Salicornia (Gco) marine algae and the as prepared nanoparticles evaluated for their anticancer potential against breast cancer (MCF-7) cell line under in vitro conditions and antibacterial activity against bacterial pathogens. This biological strategy is a cost-effective and an alternative approach to the conventional chemical and physical methods of synthesis.

Material And Methods
Extraction of β-glucan from Gracilaria corticate (Gco) The red algae (Gco) were collected from the Bay of Bengal, Mandabam, Rameswaram Tamil Nadu, India. The algae were cleaned with double distilled water to remove excess debris and were allowed to completely dry. The dried algae (50 g) were extracted with 0.1 M Hydrochloric acid (HCL) for 2 h at 60ºC.
At the end of 2 h, the algal extracts were naturalized and spun in a centrifuge at 5000 rpm. Then, the supernatant was collected and dried by using a rotary evaporator at 60ºC. The dried powder washed with MilliQ water to remove excess debris and stored at 15°C for further characterization and biological activity studies.

Synthesis of β-GluNPs
Typically, 1g of β-Glucan dissolved in 100 ml of 2 % Sodium hydroxide (NaOH) in a magnetic stirrer for 3 h at 90ºC. After incubation, β-Glucan nanoparticles (β-GluNPs) were precipitated by adding 1% acetic acid. The synthesized nanoparticles were stabilized by adding dropwise TPP solution (2 mg/ml) to the β-GluNPs (5 mg/ml) in a magnetic stirrer at room temperature. The mixture stirred for 1 h at room temperature. At the end of incubation, the solution was spun in a centrifuge at 5000 rpm and the pellet was washed with MilliQ water to remove unreacted nanoparticles and kept at 4ºC for further use.

Characterization of β-GluNPs
The characterization of β-Glucan and β-GluNPs were done by using high throughput instruments as follows 1

H NMR analysis
The synthesized β-Glu and β-GluNPs were dissolved in DMSO-d6. The dissolved sample poured into the NMR glass tube placed on the NMR JEOL. Nodel ECZ500-spectra were recorded with the 500 MHz spectrometer and the temperature was maintained at 80 ºC. The 1 H NMR spectra in the 2000 total scan range (0-7 ppm) were recorded.

FTIR Spectra analysis
The synthesised β-Glu and β-GluNPs made as thin powders and mixed with KBr to make pellets.
The pellets were analyzed on a Thermo-Nicolet 6700 -FTIR spectrophotometer in the infrared spectral range from 400-4000 cm −1 .

UV-Spectral analysis
The β-GluNPs were dissolved in deionized water and poured in a glass cuvette was placed in Shimadzu MPC3600 UV-VIS spectrometer and UV range recorded from 200-500 nm Analysis of DLS and ZETA potential for β-GluNPs The β-GluNPs dissolved in deionized water and poured in a glass cuvette. The cuvette kept on the ZETA PALS and the temperature was maintained at 25 ºC. The nanoparticle size and the distribution range polydispersity index (PDI) value was recorded.

HR-TEM and SEM analysis for β-GluNPs
The β-GluNPs desiccated for 12 h. The desiccated nanoparticles were placed on the copper grid material on the Titan Themis 300kV from FEI, ULTRA 55 -GEMINI technology. The nanoparticles size, structure, and micrograph recorded.

X-Ray Diffraction (XRD) for β-GluNPs
The desiccated β-GluNPs, placed on the glass slide. The sample glass slide kept on the Rigaku Smart Lab general purpose X-ray diffractometer system. The diffraction intensities of the nanoparticles ranging from 10º to 90º 2θ angle were recorded.

Antibacterial activity of β-GluNPs
The bacterial cultures such as Staphylococcus aureus MTCC 96, Bacillus subtilis MTCC-2387, Pseudomonas aeruginosa MTCC 424 and Proteus vulgaris MTCC 426 were procured from Microbial Type Culture Collection (MTCC), Chandigarh. The antibacterial activity of β-GluNPs evaluated by using agar well diffusion method. The gel puncture used to cut the wells around 6 mm and swabbed with 0.1 ml of bacterial pathogens at the concentration of 10 8 CFU/ml. The β-GluNPs was loaded in each wells at concentrations of 50, 75 and 100 μg/ml, respectively. The Ampicillin is used as a positive control and incubated at 37±0.2°C. After 24 h incubation, the zone of inhibition (mm) measured. The assay performed in triplicates with positive control.
in vitro cytotoxicity assay for β-GluNPs

Results And Discussion
The extracted Beta-Glucan was 11.6 g from 100 g of Gracilaria corticate. Further, the generation of β-GluNPs, β-Glucan was solubilizing in NaOH under ambient conditions. The synthesised nanoparticles were elevated by tripolyphosphate, which relied on shape, structure uniformity, and stability of the nanoparticles. The synthesis of β-GluNPs was predicted by 1 H NMR signals, which were assigned to β-Glucan and β-GluNPs and attributed to β-1, 6-linkage. The 1 H signal at 3.39 assigned to the unlinked H-6 of Glucan. The signal at 5.37 represented H-1 phosphate attributed to the α-anomer of the reducing end. The appearance of peak at 5.38 PPM due to the OH--P=O binding in spectra Fig 1B, which con rms the βglucan nanoparticle formation. The β-Glucan nanoparticles were in accordance with the signals of the similar compound reported in literature [28].

DLS and Zeta potential analysis
The hydrostatic diameter of the β-GluNPs assessed by DLS con rming the particle size to be 88.91 nm and the particles were narrow sized and highly dispersed [Poly Dispersity Index-0.225] (Figure 5a) The zeta potential was recorded as -22.7 mV that suggested the colloidal suspension of the nanoparticles. The negative effect could be due to TPP binding, which created repulsion forces on the nanoparticles (Figure 5b) [32,33].

XRD analysis
The XRD pattern depicted the diffraction peaks of the β-GluNPs (Figure 6). The peaks corresponded to 2θ=23.08º, 34.5 º, 45.2º, which were planes of crystalline nature of the GluNPs and were in accordance with the earlier reports on polysaccharides [34,35] HR-TEM analysis HR-TEM micrographs manifested the ne motifs of uniform sized β-GluNPs with a mean size of 20±5nm.
The β-GluNPs were colloidal, circular shaped, with a smooth surface and durable for a long period ( Figure  7a). SEM micrograph also revealed the circular shape and the smooth surface (Figure 7b). The well dispersed nanoparticles were observed in both SEM and HR-TEM mainly due to the presence of TPP involved in the formation of nanoparticles [36]. The HR-TEM and DLS size of the nanoparticles were variant due to the different techniques and principals involved.

Antibacterial activity
The antibacterial activity of the β-GluNPs evaluated against bacterial pathogens. The highest zone of inhibition was perceived in B.subtilis (15 mm) followed by S.aureus (12 mm) and P.aeruginosa (12 mm).The slightest activity was observed in P.vulgaris (10 mm) (Figure 8). From the obtained results, known that β-GluNPs performed excellent activity against both gram positive and gram-negative bacterial pathogens. But, when compared with Gram negative the β-GluNPs have potential antibacterial activity against gram positive bacterial pathogens, due to variance in the membrane structure thickness in grampositive and gram-negative also in uence the bactericidal activity of β-GluNPs [37,38].

in-vitro anticancer activity
The cytotoxicity effect of the β-GluNPs assessed against MCF-7 breast cancer cell line in a dosedependent manner (10,20,25,50,75, 100 and 150 µg/ml). The effect of nanoparticles at different concentrations revealed by the cell death observed and IC 50 values were 42.5µg/ml and 52.6µg/ml for β-GluNPs and β-Glucan, respectively ( Figure 9). These results proposed that the cytotoxicity e cacies of the synthesised β-GluNPs were stronger than free β-Glu, which described an increased anticancer e ciency of β-Glucan nanoform. This might have been due to the shape, structure, and surface functionalization as well as the high penetrable nature of the nanoparticles that accumulated inside the cancer cells and generated stress, which nally led to cell death [39]. Similarly, Yugay, et al. [24] reported that metal-coated beta-Glucan nanoparticles exhibited cytotoxicity against the cancer cells. The apoptosis effect due to Glucan triggered the immune system, which ultimately led to the programmed cell death and reduced tumor resistance [40,41].
The competence of β-GluNPs to induce apoptosis ascertained by AO/PI staining. Figure 11 shows the stained cells that were characterized as viable cells in light green while showing undamaged nuclear structure. Early apoptotic cells were indicated by orange uorescence while the late apoptotic cells, were shown in red color indicating the shrinking cell density with nuclei condensation and membrane blebbing in contrast. Apoptosis is programmed cell death caused by genetic and structural alterations [42,43]. The induction of apoptosis in cancer cells is the crucial characteristic of several anticancer agents.
The treated cells' DNA fragmentation pattern was analysed, to con rm that the MCF 7 cell death induced by β-GluNPs was via apoptosis and not via necrosis, The characteristic ladder pattern on the agarose gels, which is the result of the endonuclease mediated inter nucleosomal chromosomal DNA fragmentation, is the key biochemical sign of apoptosis ( Figure 12). In necrotic cell death, random fragmentation of chromosomal DNA results in fragments, which give a smear like appearance on agarose gels. β-GluNPs treatment induced inter nucleosomal DNA fragmentation at the IC 50 -85.5µg/ml concentration [44].

Conclusion
To Sum up, the β-GluNPs were found to inhibit the bacterial pathogens and trigger apoptosis in MCF-7 cells by an increase in inter chromosomal DNA fragmentation. DNA fragmentation, a biochemical hallmark of apoptosis assumed the terminal step in the apoptosis cascade. The results of the present study have provided a strong evidence on the capability of the β-glu nanoparticles to enhance genomic DNA fragmentation resulting in the typical DNA ladder pattern in treated Hela cells. All these results indicated the potential of β-GluNPs in inducing apoptosis of MCF-cells. Further studies to nd the mechanism of apoptosis of MCF-cells is under process.