Effectual Anticancer Potentiality of Loaded Bee Venom onto Fungal Chitosan Nanoparticles

Biochemistry Department, Faculty of Science, University of Tabuk, Saudi Arabia Bioinformatics Department, Genetic Engineering and Biotechnology Research Institute, University of Sadat City, Sadat City, Egypt Faculty of Aquatic and Fisheries Sciences, Kafrelsheikh University, Egypt Chemistry Department, Faculty of Science, Ibb University, Yemen Department of Nutrition and Food Science, Faculty of Home Economics, University of Tabuk, Saudi Arabia Department of Home Economics, Faculty of Specific Education, Kafrelsheikh University, Egypt Biochemistry Section, Chemistry Department, Faculty of Science, Tanta University, Egypt

Bee venom (BV) is the released defense weapon from honeybee (Apis mellifera) when their colony is attacked. BV contains composited mixtures from bioactive peptides that could protect bees' colony against a broad diversity of predators and invaders. The contained bioactive components in BV include melittin (the major BV constituent), adolapin, apamin, degranulating peptide (for mast cell), enzymes (hyaluronidase and phospholipase A2), and some nonpeptide constituents (e.g., dopamine, histamine, and norepinephrine) [10,11].
The BV or its constituents (e.g., melittin) were extensively investigated as potential treatment and inhibitors of tumor types; they exhibited multiple potential molecular anticancer mechanisms [11][12][13]. Numerous studies and reviews were documented to verify the anticancer capabilities of either BV or melittin toward various tumor types [14][15][16][17][18]; they concluded that BV with its components has auspicious anticancer and cytotoxic agents with broader spectrum toward multiple tumor cell types.
The nanotechnology involvement in most biomedical fields became much prevalent, including nanomaterial applications themselves as antimicrobial, anticancer, antiviral, and biocidal agents, or their loading with bioactive drugs/compounds to increase their solubility, stability, functionality, and delivery to the human body [19]. The nanopolymers, particularly nano-Cts and nano-FC (abbreviated thereafter as NFC), were from the most evaluated nanomaterials for their bioactivity and functionality as drug carriers, biopreservatives, and antimicrobial, anticancer, and gene delivery agents, individually or composited with other active compounds [3,[20][21][22][23].
Accordingly, current study targeted the production of NFC, its loading with BV, and evaluation of their physiognomies and bioactivities to inhibit cervix adenocarcinoma (HeLa) cells and elucidate their potential action as anticancer agents.

Materials and Methods
2.1. Fungal Chitosan (FC) Extraction. Fungal culture, Fusarium oxysporum f. sp. medicaginis, ATCC® 52169™ (abbreviated thereafter as Fom), was used herein for FC production. Broth and agar media of potato dextrose (PDB and PDA, Difco Laboratories, Sparks, MD) were employed for Fom culture propagation and maintenance. Fungal incubation in PDB was prolonged for 7 days at 28°C under agitated aerobic conditions. The FC extraction was accomplished according to Tayel et al. [6]. Briefly, after cultivation, Fom mycelia were harvested via filtration, washed by deionized water (DW), and dried at 45°C in air oven. Dried Fom mycelia were deproteinized (using 1 M of NaOH), demineralized (using 1 M of acetic acid), and deacetylated (with 58% w/v NaOH solution at 110°C for 90 min); the filtration and extensive DW washing were repeated after each step. The FC deacetylation degree (DDA) was determined from its FTIR (Fourier-transform infrared spectroscopy) spectrum, using the Baxter et al.

Synthesis of
where C i is the BV concentration in release solution at time i, V is the total release solution volume, and V s is the sample volume.

Evaluation of NP Antiproliferative Activity.
The BV/NFC-NPs antiproliferative activity was assessed using MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay [30]. In a 96-well polystyrene plate, each well contained 100 μL from complete medium, tumor cells (~1 × 10 4 ) were inoculated and incubated for 24 h in humidified air with 5% CO 2 at 37°C. After cell adherence, wells received gradual amounts of NFC, BV, and BV/NFC-NPs, after dissolving in DW, to have a concentration range of 0-500 μg/mL from each agent. After another 24 h of incubation, fresh media were added to replace the treatment solution and 10 μL of MTT solution (5 mg/mL of media without phenol red and serum) and plates were additionally incubated for further 3 h until purple formazan color appeared. After supernatant discarding, formazan crystals were dissolved with 100 μL of DMSO and the color was appraised at 540 nm absorbance within 1 h. Plain DMSO served as control.

Valuation of HeLa Cell Apoptosis Using Acridine
Orange/Propidium Iodide Combined Staining. The HeLa cell potential apoptosis, after treatment with BV/NFC nanoconjugate, was valuated via combined staining with acridine orange (AO) and propidium iodide (PI) [31]. HeLa cell suspension (~2 × 10 5 cells/mL) was treated with BV/NFC-NPs at 500 μg/mL concentration and incubated for 24 and 48 h in humidified air with 5% CO 2 at 37°C. Treated and control cells were rinsed using PBS, then double stained for 15 min with AO (10 mg/mL) and PI (4 mg/mL) in the dark. Images were captured (using fluorescence microscope Olympus BX51, Tokyo, Japan) based on the appearance of apoptosis indicators, i.e., the green-, orange-, and red-stained cells/organelles, within 25 min after staining.
2.5.4. Appraisal of Cell Apoptosis via DAPI Staining. The BV/NFC-NPs-induced apoptosis was further analyzed using nuclear dye DAPI (4 ′ ,6-diamidino-2-phenylindole) fluorescent staining to detect nucleic DNA fragmentation and condensation in treated cells [32]. The treated HeLa cells were handled as mentioned in Section 2.5.3; then, after PBS washing, they were fixed with paraformaldehyde (4%) for 12 min and permeabilized with buffer (containing 0.5% Triton X-100 and 3% paraformaldehyde), then 50 μL of DAPI (with 1 μg/mL concentration) was added and kept for 60 min. The fluorescent micrographs were captured to demonstrate the liberated and condensed nuclei fragments stained by DAPI fluorescent dye.

Results
The FC extraction and production, from Fom grown mycelia, were gainfully achieved in this work; the FC productivity was 2.41 g/L of fermentation medium. The produced FC had a molecular weight of 51.6 kDa and DDA of 88.4%.
The process effectiveness of ionic gelation to transform FC into NFC and loading it with BV was assessed using FTIR to reveal their biochemical bonding and interactions.
The FTIR spectra of FC, NFC, BV, and NFC/BV nanoconjugates are presented in Figure 1. In the spectrum of Fom-extracted FC (Figure 1, FC), the strong peak around 3452 cm −1 corresponds to the combined hydrogen bonding and O-H stretching; the N-H stretching (from primary amines) was overlapped in this region. The specified absorption peaks and bands of the FC were detected at 1718 cm -1 (carbonyl (C=O) stretching of the secondary amide I band), peaks at 1539 cm −1 and 1324 cm −1 (designated the amide II (N-H) bending vibration and the absorption of amide III, respectively), and at 1079 cm -1 (belongs to the C-O-C stretching). The spectrum of NFC (Figure 1, NFC) indicated some differences from FC spectrum; the peak of 3452 cm −1 in FC became wider in NFC with amplified relative intensity, which indicates the enhancement in hydrogen bonding. The NFC spectrum showed also a sharp P=O peak at 1174 cm-1, indicating the cross-linkage with TPP. Generally, the main characteristic peaks and bands in FC spectrum appeared in NFC spectrum, but with slight shifting of them to close wavenumbers.

International Journal of Polymer Science
For the BV spectrum, the observed peak at absorbance region of 3250-3450 cm -1 indicates the free vibrations of N-H stretching. The FTIR spectrum of BV also indicated the characteristic amide bands, i.e., amide I (1651 cm -1 ), amide II (1532 cm -1 ), and the bands at 1112 cm -1 and 1040 cm -1 that indicate unsystematic coil conformation (Figure 1, BV). In the FTIR spectrum of NFC/BV nanoconjugate, the BV absorbance peak for N-H stretching at 3318 cm −1 was reduced and shifted to 3421 cm −1 (Figure 1, NFC/BV). The amide I indicated band in the BV at 1651 cm −1 was also shifted to a higher wavenumber (1654 cm −1 ) in BV/NFC.
The characteristic attributes of synthesized NP from FC and BV are illustrated in Table 1.
The NFC had smaller average (mean) particle diameters, median diameter, and size range than BV/NFC nanocomposite. The PDI values were <0.5 for both synthesized NPs (0.262 for NFC and 0.414 for BV/NFC nanocomposite), which indicates a matched and favorable NP size distribution (Table 1). Both NP types had positive Z-potentialities; the BV loading into NFC slightly reduced their positivity from +33.7 (for NFC) to +27.2 (for BV/NFC nanocomposite).
The particle shape, size, and distribution, for BV/NFC nanocomposite, were further elucidated via TEM imaging ( Figure 2). The nanoconjugate particles mostly appeared with spherical shapes and smooth surfaces, uniform distribution with size range of~168-256 nm (Figure 2).
The calculated LC and EE, of BV in NFC, were 38.22 and 92.42%, respectively. The BV release from NFC was in vitro evaluated at pH 5.2 and 7.0, through direct dispersion during 30 h of release duration. The BV release patterns from NFC are plotted in Figure 3.
The BV release had a pH-and time-dependent manner; lower BV percentages were released in pH 7.1 than in pH 5.2. The releasing pattern revealed a burst BV release at the first 6 h; after that, gradual releases were detected with experiment prolongation for 30 h. The released BV ratios were 67.2 and 71.4% after 6 h, and the maximum release percentages were 76.2 and 81.3%, at the duration end, in pH 7.1 and5.2, respectively.
The anticancer potentialities of NFC, BV, and FC/BV-NPs measured with the decrement of HeLa cell viability, after exposure to different concentrations from synthesized agents, are plotted in Figure 4. The entire agents had potent anticancer activity toward examined cells; the activities of all agents were dose-dependent. The most forceful agent was the NFC/BV nanoconjugates then the BV and lastly the NFC. After 24 h of exposure to a concentration of 500 μg/mL, only 8.3 and 22.3% of cells remained viable after treatment with NFC/BV-NPs and BV, respectively. The 200 μg/mL concentration from NFC/BV-NPs could kill 50% of cancerous cells after 24 h (Figure 4).
Fluorescent imaging was applied to elucidate the cytotoxic effects of NFC/BV composites toward examined cancerous cells ( Figure 5). The potential apoptotic effect after HeLa cell treatment with NFC/BV nanoconjugates (at 500 μg/mL concentration) was investigated using DAPI staining ( Figure 5, DAPI). The exposure to nanoconjugate induced observable morphological apoptosis signs in treated HeLa cells, e.g., membrane blebbing, cell shrinkage and rounding.
The apoptosis signs greatly increased and became more vigorous, with exposure prolongation from 24 to 48 h, to include most of the treated cancerous cells. While control cells had no apoptosis signs, the DAPI-stained treated cells had many brightly fluoresced fragments/nuclei; the DNA condensation and apoptosis signs increased dramatically in a time-dependent approach.
The cell apoptosis and necrosis were further elucidated via AO/PI double fluorescent staining ( Figure 5, AO/PI); viable cancerous cells appeared with intact structures and green color for cells and nuclei. The early apoptosis signs include the appearance of chromatin condensation and bright green-stained nuclei, which clearly appeared after 24 h from treatment with NFC/BV nanoconjugate. The secondary necrosis and late apoptosis signs evidently appeared in HeLa cells after treatment for 48 h, with more chromatin condensation, dense orange-stained areas, and many stained nuclei with deep reddish-orange color.

Discussion
The achievement of FC was successfully conducted from Fom mycelia, which confirmed the investigations that indicated the high potentiality of fungi as sustainable sources for   International Journal of Polymer Science chitosan production [4-9, 22, 23]; these investigations recommended the application of FC, from many fungal species, in the biomedical and health-related fields.
The FTIR analysis of FC certified its comparable structure/groups to the standardized conventional chitosan [6,33].
The slight shifting of many characteristic peaks and bands in FC spectrum to close wavenumbers in NFC spectrum validates the successful NFC cross-linkage and synthesis of targeted NPs [22]. The NFC and NFC/BV spectra were closely matched in most of the biochemical groups and wave-number intensity; this indicates that BV did not chemically affect or change the NFC structure and accordingly maintain its biosafety for usage in the human body [29]. In the FTIR spectrum of NFC/BV nanoconjugate, the stretching and broadening of C-O (from NFC) and reduction of stretched N-H intensity (from BV) and their shifting to littler wavenumbers indicate the hydrogen bond formation between these active groups from BV and NFC [34].
The obtained data from "Zetasizer," regarding the synthesized NFC and BV/NFC nanoconjugate, revealed that BV/NFC nanoparticles had larger sizes than NFC particles; this could conceivably be due to the added molecular weight and composited structure after venom on nanochitosan [27], which verifies the elevated ability of current NFC to carry BV. For successful biomedical applications, functional nanocomposites are mostly manufactured in a NP size range from 20 up to 300 nm, considering that the "smallest particles could reach any part of the body" [35].
The BV release from NFC was very fast in the first 6 h of the experiment; this burst release is supposed to depend on the BV dissociation from the surface of NFC, as generally reported from loaded protein molecules on nanochitosan [21,36]. Additionally, the early rapid release and diffusion of protein molecules from the surface of nanoparticles were stated as undeniable [37]. Subsequently, the slow BV release was seemingly attributed to slow degradation of entrapped protein molecules in NFC and the degradation of     5 International Journal of Polymer Science nanoparticles itself [38]. The degradation rate of protein was supposed to exceed its releasing rate, after prolonged releasing period [39]. Additionally, BV was reported to engage the long chain of nanochitosan in a nonuniform manner, thus high percentages from it could be easily released in the early releasing period [28].
The release of BV was higher in pH 5.2 than in pH 7.1, which could be explained by the solubility preference of chitosan in weak acidic solutions than in neutral solutions; thus, the pH 5.2 condition could increase BV liberating from the solubilized NFC.
The induction of HeLa cell apoptosis/necrosis, after treatment with BV/NFC nanoconjugate, was evidenced via staining with fluorescent dyes. The lethal effect of BV/NFC nanoconjugates could be principally owed to BV anticancer potentiality, which was strengthened after loading onto NFC. The NFC polycationic nature is suggesting for facilitating particle attachment to tumor cells and enables more contact between BV and cell surface.
Animal venom peptides, especially BV, have a great potential for developing into biopharmaceuticals, with many limitations that could be overcome with their conjugation with polymeric nanocarriers like Cts to augment their chemical instability, half-lives, oral absorption, and target cytotoxicity [40].
Although the produced bioactive agents (NFC and BV) were extracted from fungal and insect sources, the transability of other biological components from these organisms to the human body is not expected because Cts and NFC are mostly polysaccharides extracted with harsh steps (e.g., very high alkalinity and temperature), leading to the elimination of any protein residues, while the BV is obtained from the bees' toxin glands and is anciently applied for treating various diseases with no evidences for genetic transferability during these treatments [13]. Nanoformulations are demon-strated as well-suited for the administration of drug-like molecules; polymeric nanocarriers can enhance the drugs' half-life circulation and their deposition into diseased sites with diminished extravasation to healthy/normal tissues [41].
The BV antitumor activity was stated to depend primarily on close contact between its active constituents and cancer cells, which is essential to develop cell apoptosis/necrosis [42]. Melittin (the major protein constituent of BV) was confirmed for cancer therapy as a potent bioactive agent [43]; the main suggested functions of melittin involved cell membrane perturbing (leading to hemolytic and antimicrobial consequences) and the induction of vigorous structural alterations in these membranes (including pore formation, vesiculation, and fusion) [44]. Furthermore, melittin was verified to induce arrest in tumor cell cycle, inhibiting their growth, and apoptosis signs in different tumor cells [12]. Interestingly, BV melittin was demonstrated to specifically pursue cells that have extraordinary levels from Ras oncogene, i.e., tumor cells [45].
Apoptosis was appointed as the key function of BV anticancer action, as consequences from death receptor (DR) stimulation and inactivation of nuclear growth factor family kappa B (NF-κB) in cancer cell [11].
The application of AO/PI double fluorescent staining, for quantifying the apoptotic and necrotic consequence of BV/NFC nanoconjugate on HeLa cells, proved its applicability and efficiency. The variations in cell staining with green, orange, and reddish-orange as indicators for early apoptosis, late apoptosis, and secondary necrosis, respectively, were illustrated [31]. The tumor cell death indicators (apoptosis, necrosis, and lysis) were stated as the potential mechanisms, by which BV could inhibit tumor development [17]. A previous study [46] indicated that BV anticancer activity was more forceful against V79 than for HeLa cells; and its effect toward both cells was dose-dependent. The BV  International Journal of Polymer Science role in the in vivo inhibition of cancer growth and proliferation was addressed to involve immune response stimulation of lymph nodes [42]. Interestingly, BV was illustrated to induce leukemia cell apoptosis, with no cytotoxicity on normal cells of the bone marrow [15]; the main regulators of BV-induced leukemic cell apoptosis are caspase-3 and Bcl-2 via suppression of mitogen-activated signaling paths [14]. The preceding flow cytometric analysis indicated BV capability for inducing ROS (reactive oxygen species) production, increasing cytoplasmic Ca 2+ levels, releasing cytochrome C due to reduced potentials of mitochondrial membrane, and promoting caspase-3 activation, which consequently caused cell apoptosis [16].
The amount of BV binding sites from carbohydrate/amines on cancerous cell membranes was suggested to explain the varying sensitivity of cancer types to BV components [46]; thus, it is supposable here that loaded NFC with BV could generate more binding sites with HeLa cell membranes and accordingly strengthen the BV antiproliferative action toward these cells.
The modern approaches for NP safety assessment recommended the usage of in vitro/ex vivo models as essential prerequisites for their translation into applicable products [47]. The Safe-by-Design (SbD) concept is applied for the assessment of nanomaterial and nanopolymer potential consequences on the environment and human health and their uses to develop nanomedicines; according to the latest SbD criteria, NFC could be regarded as an ideal safe "nanobiocarrier" for delivering BV into the human body to fight cancer cells [48,49].

Conclusion
The grown mycelia of Fom were innovatively employed as FC source, which was successfully utilized for NFC production and loading with BV. The BV/NFC nanoconjugate had favorable structural and biochemical physiognomies, plus their augmented anticancer bioactivity against cervix carcinoma (HeLa) cells. The BV/NFC nanoconjugate could induce serious apoptosis signs in HeLa cells in a time-and dose-dependent manner. The application and extra evaluation of BV/NFC nanoconjugates are strongly recommended as effective and natural anticancer agents.

Data Availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest
The authors declare that they have no conflicts of interest.