I. Isolation and safety pattern of wild mushroom polysaccharides.
- Isolation and purification of wild mushroom strains.
Wild mushrooms growing species (9 isolates; I1-I9) were collected from Bahira, Egyptin in sterile plastic bags. In a sterile hood, the gills of the fruiting bodies were cut into small pieces and transferred into Potato Dextrose Agar medium (PDA) plates. The inoculated plates were incubated at 25°C for 5 days; fungal purification processes were performed on PDA media by subsequently subcultures then, the purified strains were stored in 40% glycerol at -80°C.
- Extraction of mushroom polysaccharides.
Mushroom polysaccharides samples (I1-I9) were extracted according to Wu method (Wu 2017), the filamentous hyphae of the purified fungal strains were cultured on Potato Dextrose broth medium at 27°C for 8 days. After the formation of mycelial beads, the culture supernatants were collected by centrifugation at 4000 rpm for 15 min. The collected supernatants were boiled at 90°C for 1 hour, and the polysaccharide samples were overnight precipitated using absolute ethanol at 4°C. The crude polysaccharides were recovered by centrifugation at 5000 rpm for 30 min, and finally the resulted powder samples were dried in oven at 50°C for 48 hrs.
- Safety assay of the extracted polysaccharides.
For the determination of polysaccharides (I1-I9) nontoxic concentration, the cytotoxic assay was performed on peripheral blood mononuclear cells (PBMCs) using MTs assay Kit. Briefly, PBMC were isolated from blood samples of healthy volunteers using Ficoll-Hypaque density gradient centrifugation protocols, the recovered cells were seeded at concentration 6×104 cells/ml in 96-well plates (100 µl cell suspension per well). After 24 hours, 100 µl of the polysaccharide concentrations were incubated with PBMC for additional 48 hours. At the end of incubation, PBMCs cellular viability was quantified using BioVision's MTS Cell Proliferation Assay Kit. The mushroom strain that produced the safest polysaccharide sample was selected for molecular identification.
The polysaccharides samples which showed the lowest toxic pattern on PBMCs was selected for molecular identification, microencapsulation and testing its activation effects on NK cells.
- Genotypic Identification for the selected strain using 18S rRNA sequence.
Fungal DNA was isolated using Fungal DNA/RNA/Protein Kit (Qiagen) according to the instruction protocols. Two universal primers, forward primer (5’-ATCTGGTTGATCCTGCCAGT-3’), and reverse primer (5’-GATCCTTCCGCAGGTTCAC3’) were used for the amplification of DNA regions of the mushrooms18S rRNA gene. The purified PCR product was subjected to DNA sequencing using forward primer in the sequence reaction. Sequencing was performed using Big Dye® Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) and model 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The obtained DNA nucleotide sequences were analyzed using NCBI-BLAST for confirming the identity of the obtained sequences (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple sequences alignment of the sequences and the other published ones were performed using ClustalW (1.83)(Thompson JD 1994). The amino acid sequences were used for comparison using MEGA 4 (Tamura K 2007), and phylogeny was tested with bootstrap method with 2,000 replications. The phylogenetic tree was analyzed and generated based on UPGMA statistical method.
II. Micro-encapsulation efficacy of the selected polysaccharides of the identified wild mushroom strain.
- Micro-encapsulation process of the selected polysaccharide
Microencapsulation process of the selected mushroom polysaccharide was performed using the method of Mohamadnia et al. (Tamura K). Sodium alginate (Alg; with a viscosity of 15,000–20,000 centipoises (cps)), κ-carrageenan, and calcium chloride anhydrous were purchased from Sigma-Aldrich (Schnelldorf, Germany). Alg and κ-carrageenan solutions were prepared separately by dissolving the biopolymer in distilled water followed by heating at 70°C and 80°C for Alg κ-carrageenan, respectively for 30 min. Polysaccharides (100 mg) were dissolved in distilled water (2.5 ml) and mixed with 5 ml of 0.5 % Alg and 2.5 ml of 1 % κ-carrageenan solution (pre-sterilized at 121°C for 15 minutes). An Inotech Encapsulator IER-50 (Switzerland; equipped with a 100µm/300 µm concentric nozzle and 20 ml syringes to pump the Alg/κ-carrageenan*polysaccharide solution) was used to produce microcapsules in voltage range 400—1700; schematically illustrated by Mahdavinia et al. (Mahdavinia et al. 2004) (Fig. 1). Microcapsules were produced at optimum solution flow rates, vibration parameters and electrostatic dispersion settings. The resulted microcapsules were incubated in a hardening solution of 3% CaCl2 with continuous stirring for 30 min. Finally, the Alg/κ-carrageenan*polysaccharide microbeads were rinsed twice with water and dried on tissue paper.
- Characterization of polysaccharide and Alg/κ-carrageenan*polysaccharide microcapsules.
The morphology of polysaccharide, Alg/κ-carrageenan microcapsules, Alg/κ-carrageenan*polysaccharide microcapsules were investigated by SEM “JEOL, JSM-6360LA, Japan” at an acceleration voltage of 10.0 KV at different magnification powers. The FT-IR spectroscopy was measured via Shimadzu FTIR-8400 S Fourier transform spectrometer. Thermo-gravimetric analyses (TGA) for all formulations were carried out at a temperature range from 25 to 600°C under nitrogen atmosphere with heating rate of 10°C /min using Shimadzu TGA-50, Japan. Raman spectra of the samples were obtained via Raman Microscope (Bruker, SenterraII, Germany) with the following parameters: power, 200–250 mW; scans, 100–1500; resolution, 4 cm-1; crack, 5 mm; and wavelength, 1064 nm.
Polysaccharide in vitro release from Alg/κ-carrageenan*polysaccharide microcapsules.
In vitro polysaccharide release from the 100 µm microcapsules was carried out in the simulated gastrointestinal fluid at pH 1.2, phosphate buffer saline (PBS) pH 7.2, and acetate buffer solutions pH 3.6, 4.6 and 5.6. The Alg/κ-carrageenan*polysaccharide microcapsules were suspended in 50 ml of dissolution media at different pH values. These dissolution media were stirred at 120 rpm in a horizontal laboratory shaking water bath at 37 ± 0.5°C at different time intervals. Aliquots of the used buffer were withdrawn (replaced with same volume of fresh buffer to maintain the constant volume) to calculate the concentration of the released polysaccharide using phenol sulfuric acid assay (Cuesta G 2003). All release tests were performed in triplicate.
- Swelling behavior of Alg/κ-carrageenan*polysaccharide microcapsules.
Swelling behavior of the Alg/κ-carrageenan*polysaccharide microcapsules were evaluated at pH 7.4. The microbeads were accurately weighed and suspended in 10 ml of buffer solution. After specific time interval, the swollen beads were recovered and weighed again. The swelling ratio was calculated by the following equation (El-Aassar MR):
Swelling ratio (%) = (Ws - Wd)/ Wd×100……………………..(El-Aassar MR)
Where Ws and Wd are the weights of swollen and dry beads, respectively.
III. The Effects of Alg/κ-carrageenan*polysaccharide microcapsules on activating NK cells against colon cancer.
- Natural killer cells
Natural killer cells were isolated from blood samples of health volunteers using RosetteSep™ Human NK Cell Enrichment Cocktail Kit. The Kit used targets non-NK cells for removal with antibodies recognizing specific cell surface markers.
- NK cells activation using Alg/κ-carrageenan*polysaccharide microcapsules.
After NK cells isolation, the recovered cells were culturing in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS) and 1 % penicillin-streptomycin solution at 37°C in 5% CO2 for 24 hours with prepared monocytes as feeder cells (1:2 ratio; NK: feeder cells) (El-Deeb NM 2019). After incubation, the exhausted old medium was discarded and replaced with Alg/κ-carrageenan*polysaccharide microcapsules (1 ml) suspensions or RPMI medium (as a negative control), cells were incubated for 36 hrs for activation.
-NK cell- CD activation markers quantification after Alg/κ-carrageenan*polysaccharide microcapsules treatment.
After NK activation with Alg/κ-carrageenan*polysaccharide microcapsules (ANK cells), cells were collected for the activation marker quantification using monoclonal antibodies against CD56, CD16 and CD 11b (BD bioscience) at 1:400 in FACS blocking buffer, the data were acquired using calibur flow cytometry.
IV. Anticancer effect of Alg/κ-carrageenan*polysaccharide microcapsules-activated NK cells (ANK cells) against colon cancer.
- The effect of activated NK cells on CaCO-2 Cell Lines
The anticancer effects of the ANK cells on colon carcinoma cells (CaCO-2cells) were quantified by using MTS assay and compared with NK cells that activated with polysaccharides (5 mg/ml). Both activated NK cells were incubated with CaCO-2cells in a co-culture model at 37°C in 5% CO2 for 2 days with ratio (1:2, CaCO-2: NK cells). After 3 days, the exhausted culture medium was discarded, and the cytotoxic inhibition percentages on cellular viability were quantified by BioVision's MTS Cell Proliferation Assay Kit.
- Cell cycle analysis
The cell cycle pattern of CaCO-2- ANK treated cells was determined using Propidium Iodide assay. Aliquot of 6×104 cells/ml CaCO-2 cell suspension was cultured in 6 well cell plates that were incubated for 24 hours at 5% CO2 and 37°C. After semi confluency, the exhausted media was removed and replaced with 2 ml of ANK cells at ration 1:2. The treated cells were incubated at the same previous conditions for 24 hours. After incubation, cells were collected, fixed in ethanol and washed twice in ice cold PBS, then resuspend as 200 μL of 2 x 106 of cells in 300-500μl PI/ triton X 100 staining solution (1000μl of 0.1 % triton + 40μl PI + 20μl RNAse). After that, the cell suspension was incubated at 37°C for 15 minutes then transfer to ice. The acquire cell cycle data was recorded using calibur flow cytometry.
- Molecular Mode of anticancer action of ANK cells against colon cancer cells
The anticancer effects of the activated ANK cells against CaCO-2 cancer cells were molecularly studied. To that end, the expression patterns of CaCO-2cells oncogenes and tumor suppressor genes (Iκapα, BCL2, Survivn and TGF) were quantified using designed primers that listed in Table (El-Aassar MR). CaCO-2cells were cultured in 12 well plates (6 × 103cell/ml) for 2 days with ANK cells or polysaccharide (5mg/ml)-activated NK cells. After incubation, cells were subjected to RNA extraction using QIAamp RNA kit, the first-strand cDNA was synthesized using oligo-dT primer and the M-MuLV reverse transcriptase (Vivantis technologies, Malaysia). The house-keeping β- Actin gene was used as internal controls for standardization of the PCR product and the RT-PCR was done with the cDNA using the Eva Green dye and Light Cycler fluorimeter (BIO RAD S1000 Tm thermal cycler).
V. Statistical analysis
The experimental results are expressed as mean ± SD (standard deviation) for three replicates. The statistical analyses were carried out using graph pad prism8. Data obtained were analyzed statistically to determine the degree of significance between treatments using one- and two-way analysis of variance (ANOVA) at P≤ 0.005. Also, IC50 values were carried out using GraphPad prism 8.
VI. Ethical statement
All experimental protocol which involved human samples in the current study was approved in accordance with guidelines of the ethical committee of City of Scientific Research and Technological Applications; In additional to the ethical committee of Al-Azhar University, Cairo, Egypt.