الفهرس 
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Abstract
Aim of the Study:
The present study aimed to investigate the therapeutic effects of the venoms extracted from Apis mellifera and Vespa orientalis, loaded on a convenient nanocarrier on the activity of NF-κB in vitro against liver and breast cancer cell lines.
Experimental Methods and Results of the Study:
Venom Collection and Purification:
Carniolan hybrid Egyptian forager honey bee workers (Apis mellifera carnica); and oriental hornets (Vespa orientalis) were employed in the present study. The selected insect colonies were in the age group of 25 – 45 days and were kept in Langstroth’s hives. The adult honey bee workers were allowed to feed freely, mostly on pollens (from maize) and nectar (from cotton flowers). The adult hornets were allowed to feed on honey and nectar from selected nearby honey bee rearing cages. The rearing and venom extraction for the honey bees was carried out during the active period from June (2018) to October (2018); while for the oriental hornet, it was performed from September (2018) to early November (2018) to ensure the best potency and collection yield.
The venom collection process employed the electric shock method which was based on the principle of intermittent pulse oscillation and the characteristic, mostly aggressive, biology of stinging Hymenoptera. The effective operational voltage was 12 V, which produced an electric shock that agitated the insect causing it to attack the device and, consequently, deposit venom droplets from its sting into the plastic sheet situated on the glass plate of the device.
Upon drying, the venom deposited on the plate was taken immediately into a dark room to avoid its photooxidation, then it was scrapped off the plate using flexible blade. The collected venom powder was weighed and washed with 1:1 acetonitrile : distilled water containing 0.1% (v/v) trifluoroacetic acid to solubilize the peptides, afterwards it was centrifuged to remove insoluble materials; then it was stored at (-5oC) for later use.
Each collection cycle lasted for 30 min at a time so as not to weaken the colony. The collection process lasted for 10 days/month for 3 h/day. In each collection cycle, the feeding of the studied insect colonies was ensured. Furthermore, after each collection setting, the collector was disassembled, cleaned, and then reassembled for a new collection cycle.
The collected venoms were applied to a handmade filtration column that was pre-equilibrated with 25 mM Sephadex LH-20 gel Tris–HCl buffer (pH 6.5) containing 0.1 M NaCl. Elution was performed with the same buffer at a flow rate of 0.1 ml/min, and fractions were collected every 10 min.
Formulation of the Nanogel Carrier Particles:
Due to their favorable characteristics like being biodegradable, biocompatible and non-toxic, polyacrylic acid (PAA) and chitosan (CS) were regarded as ideal candidates, as a polymer and a copolymer respectively, for the formulation of the nanogel carrier for the venoms used in the present study.
Preparation of Chitosan:
The CS was prepared from the cuticle of the oriental hornet due to its convenient large size. The hornets were starved for 48 h before initiating chitin extraction, afterwards they were sacrificed by freezing for 24 h. The collected exoskeletons were then placed in nylon bags and frozen overnight. Approximately 10 gm of crushed exoskeleton samples were settled on foil paper and weighed. Afterwards, the samples were oven-dried for 4 days at 65°C until a constant weight was reached.
Extraction of chitin and chitosan:
The dried exoskeletons were treated by boiling in NaOH for 1 h to dissolve the sugars and proteins, thus isolating the crude chitin. Afterwards, the exoskeletons were washed with deionized water before being crushed to 0.5-5.0 mm pieces.
Demineralization:
The ground exoskeletons were then demineralized with 1% HCl overnight. Afterwards, the samples were treated with 2% NaOH solution for 1 h to break down the associated proteins. The residual chitin was then washed with deionized water.
Deacetylation:
The process of deacetylation was performed by adding 50% NaOH to the chitin samples and then boiling them at 100°C for 2 h. Afterwards, the samples were allowed to cool down before being washed with 50% NaOH and filtered in order to retain the CS. The CS was then oven dried at 120°C for 24 h.
The process of deacetylation of chitin to CS was confirmed by adding 5ml of I2 / KI solution to the CS sample, resulting in yellow color of solution. The change of solution’s color from yellow/ brown to dark purple indicated the presence of CS.
The degree of deacetylation, XD, is the parameter that indicates the molar percentage of monomeric units that have amino groups and vary from 0 (chitin) to 100 (fully deacetylated chitin). The degree of deacetylation was estimated using CHN elemental analysis and substituting in the following equation:
X_D=100 .(4-0.583093×W_(C⁄N))
, where WC/N is the mass ratio between carbon and nitrogen present in the CS sample.
The CHN analysis revealed the percentages of N, C and H, in the prepared CS sample to be 7.6, 40.4 and 7.42%, respectively. Subsequently, when substituting in the formula used to calculate the degree of deacetylation (XD), it was revealed that the prepared CS showed a 90.04% deacetylation degree, which was considered to be an acceptable percentage for the CS to be used as a copolymer in the formulation of a nanogel.
Preparation of the Chitosan-Polyacrylic Acid Hydrogel Polymer:
The polyacrylic acid - chitosan (PAA-CS) hydrogel composite was prepared while maintaining a temperature not exceeding 70oC to prevent the denaturation of the peptide components of the used bee and hornet venoms.
Purified CS dissolved in 1% acetic acid solution was introduced to AA aqueous solution. The polymerization involved preheating distilled water to 70°C in a 250-ml standard three-necked flask. At this temperature, nitrogen was introduced to remove any dissolved oxygen, then a solution of 0.66 mM K2S2O8 was added dropwise into the CS-AA mixture, to produce free radicals, while stirring with a magnetic stirrer, to initiate the polymerization reaction. During the preparation of venom-loaded hydrogel, a saturated solution of each venom was introduced with the CS-AA mix before initiating polymerization with K2S2O8.
After complete polymerization was reached, the polymer solution was filtered and added DROP wise to 1 M NaOH aqueous solution, whereupon the polymer precipitated and was neutralized to pH = 7. Afterwards, the polymer solution was poured into 15 cm Petri dishes and oven-incubated for 48 h at 60oC for dehydration to produce polymer ring films.
Each of the resulting polymer films (Bare or venom-loaded) was rod-milled thoroughly using a home-made rod mill, then sonicated and centrifuged to remove insoluble materials. Finally, the polymer films were dried to a constant weight at 50°C in 15 cm Petri dishes.
The resultant hydrogels were labelled as follows: a non-loaded hollow or bare nanospheres (BNPs); bee venom-loaded nanospheres (BVNPs); and wasp venom-loaded nanospheres (WVNPs).
Nano-formulation and Purification of the Hydrogel Polymer:
The nano-formulation and encapsulation of the extracted venoms into the polymer matrix was accomplished through nano-emulsification and solvent exchange procedures. Each of the polymers (hollow and venom-loaded) were mixed in a (50:50) solvent of water and palmitic acid. Afterwards, this solution was poured into 5% aqueous poly vinyl alcohol solution, to produce oil in water (o/w) emulsion which caused immediate sub-micronization of the polymeric particles. This emulsification was aided by exposure to a high-energy ultrasonic device.
A step specific for venom-loaded polymer particles (BVNPs and WVNPs) only was accomplished to confirm that the maximum venom payload was reached. This step involved incubating each of the venom-loaded particles in a saturated solution of the extracted venoms overnight before the precipitation and purification of VLPs.
The venom loaded matrices and blank polymer were dissolved in 1 M NaOH until a clear solution was observed just after ethyl acetate was added, which led to precipitation of a powder product (sodium salt - polymer matrix). The formed powder was washed thoroughly using 1 M HCl. The resultant nanospheres were centrifuged and re-suspended in distilled water to remove the solvent and any traces of free venoms in the upper aqueous phase (in case of venom-loaded polymers). Finally, the nanospheres were powdered by freeze-drying (-44oC). This method namely, nano-formulation via emulsion solvent diffusion (ESD) was reported to produce small, monodispersed nanoparticles with high encapsulation efficiency and reproducibility.
For further purification, the resultant nanoparticles were dialyzed using cellophane-based dialysis membrane for 24 h against distilled water to remove any unreacted molecules. The dialyzed particle dispersion was condensed and then dried to retrieve the nanoparticles.
Characterization and Drug-Polymer Biocompatibility:
A number of characterization techniques were used to shed light and elaborate on the different structural, compositional and morphological features of the formulated nanoparticles.
The utilized characterization techniques further served to confirm the major structural components of the loaded venoms, in addition to thoroughly perceiving the interactive, physical and chemical properties of the carrier nanoparticles with the loaded venoms.
Determination of the Surface Chemistry:
A sample of each prepared nanogel (BNPs, BVNPs and WVNPs) was suspended in ethyl acetate and sonicated for 30 min (42 KHz) prior to being loaded on a TEM sample grid. Afterwards, the TEM grids were left to dry in air, and no stains were applied.
Transmission Electron Microscopy (TEM) was used to determine surface morphology and texture, topography, tomography and average size of the resultant particles, in addition to providing valuable data regarding the interaction between the prepared nanogel and its venom payload.
The TEM illustrated the size distribution of the resultant nanoparticles, either hollow or venom-loaded, and clarified that the formulated hollow nanogel particles were in the size range of 17.7 to 29.1 nm in diameter, while the loaded nanoparticles were in the size range of 22.1 to 39.7 nm, and both types of formulations showed a spherical morphology. In addition, the venom payload in venom-loaded particles was confined in the center of the nanospheres (as evidenced by the change in core coloration), without any evidence of surface deposition of venom.
Dark Field Electron Diffraction (DFED) technique was used to confirm the crystalline properties of the nanogel particles when compared with the pure venoms extracted either from the honey bee or from the oriental hornet. This technique served to validate the used formulation method of the nanogel particles based on their resultant properties.
Based on the DFED ring patterns, the crystalline nature of the BV, WV, BNPs, BVNPs and WVNPs was elaborated by the appearance and disappearance of diffraction dots. Both BV and WV showed a crystalline nature, while BNPs, BVNPs and WVNPs showed amorphous characteristics.
Determination of Drug Interaction and Biocompatibility:
Fourier Transform Infrared (FT-IR) Spectroscopy was employed to interpret the chemical composition and functional groups of the nanogel particles, revealing any surface interactions, either chemical or physical. The FT-IR was recorded in the range of 400 – 4000 cm-1 using FT-IR spectrophotometer.
To assess the presence of any interactions, either chemical or physical, between the loaded venoms and the grafted CS-PAA polymer, the IR spectra of the previously mentioned specimens were compared together.
The comparative FT-IR data obtained from the PAA, CS and CS-PAA samples showed that the chemical composition of the CS-PAA copolymer was found to be nearly identical to that of PAA, indicating that CS was most-likely involve within the inner core structure of the BNPs.
Furthermore, the FT-IR data between BNPs and the free and nanogel-conjugated venoms (BV and BVNPs; WV and WVNPs) showed that the chemical structure of the BNPs was similar to that of the BVNPs and WVNPs, hence strongly suggesting that the interaction between the BNPs and their venom payload was strictly physical, most probably by encapsulation, without any evidence of chemical interaction between the loaded venoms and the nanogel carrier.
X-ray Diffraction (XRD):
The XRD technique was also used to determine the crystallinity degree, size of the crystallites, chemical nature of a compound, and type of crystalline phase. The used X-ray Diffractometer was equipped with Cu-Kα as a radiation source (λ = 1.54Å) at 40 Kv, 35 mA, 25oC and scanning speed of 0.02o/sec. The diffraction peaks were recorded between 2o and 60o 2 θ.
The comparative XRD analysis between bare BNPs, BVNPs and WVNPs indicated that both BVNPs and WVNPs had intensity peaks similar to those in the BNPs, in addition to having additional intensity peaks, most probably attributed to the aromatic compounds present in each venom payload, namely bee and hornet venoms.
This further confirms the previously detected FT-IR analysis observations, that the resultant venom-loaded nanogel particles possess venom payload encapsulated within their cores, in a strictly physical interaction without any chemical bonding formed between the venoms and their carriers.
Cytotoxicity Assay of the Extracted Venoms and the Formulated Nanogel Particles:
Sample Preparation for Cytotoxicity Assay:
The lyophilized venoms (BV and WV) in addition to the nanogel particles (Bare and venom-loaded) were dissolved in distilled water to produce the concentrations required for the cytotoxicity assay against the cell lines (HepG2, MCF-7 and WI-38).
Choice of the Cancer Model Cell Lines:
Both the hepatocellular carcinoma cell line (HepG2) and the human breast cancer cell line (MCF-7) were described to be perfect models for studying liver and breast diseases, respectively, especially cancer therapeutics, because of its high degree of morphological and functional differentiation.
The MCF-7 cell line is a human breast cancer model cell line with estrogen, progesterone and glucocorticoid receptors. MCF-7 cells were reported to be ideal for in vitro breast studies since they retained numerous ideal characteristics specific to mammary epithelium.
The WI-38 cell line is a diploid human cell line was isolated by Leonard Hayflick in the 1960s from the lung tissue of a 3-month-gestation aborted female fetus. The WI-38 cells have a fibroblast-like morphology and has been extensively used in scientific research and vaccine production. The WI-38 cell line was used in the present study as a model system for evaluating the safety of the formulated nanogel carrier and whether it posed cytotoxic effects against normal cells, either in its bare form or when loaded with the extracted venoms.
The Cytotoxicity Assay:
To evaluate the cytotoxicity of the different extracted venom formulations (free venom and venom-loaded nanogel particles) on human liver cancer (HepG2), breast cancer (MCF-7), and normal fibroblast (WI-38) cells, through their mitochondrial dehydrogenase activity, a colorimetric MTT test [3-(4,5-dimethyldiazol-2-yl)-2,5- diphenyltetrazolium bromide] was carried out.
The cytotoxicity assay involved the HepG2, MCF-7 and WI-38 cell suspensions to be evenly distributed in transparent flat-bottom 96-well micro titer ELISA plates, to study the effects of the different applied free venoms (BV and WV) and the bare (BNPs) and loaded (BVNPs and WVNPs) formulations on cell viability. The plates were then stabilized by incubation at 37˚C in a humidified (80% RH) 5% CO2 atmosphere for 24 h to assure the attachment of cells. The growth medium was then replaced and 10 µl of different concentrations of the prepared toxicants were introduced to each well. The micro-titer plates were then incubated for 48 h for the treatment to take effect, then fresh media was added to each well in addition to a total amount of 20 µl MTT reagent. The cells were further incubated for 4 h at 37 °C with 5% CO2.
Finally, the plates were read at the absorbance of a 570-nm wavelength by a 96-well microplate reader to obtain the number of viable cells relative to the control population. The results were in the form of optical densities (ODs), which were used to calculate the mean percentage of viability of the cells in each cell line for each applied toxicant. For each compound tested, the IC50 was generated from the dose-response curves for the cell lines using SigmaPlot software.
The cytotoxicity assay showed that the highest cytotoxicity values (in terms of IC50) were exhibited by free BV, followed by free WV, while the lowest cytotoxicity was always shown by the BNPs. However, the cytotoxicity of the BVNPs and WVNPs was variable among the studied cell lines, where against HepG2, BVNPs showed a higher cytotoxic effect when compared to the WVNPs; while against MCF-7 cells, the opposite was true, as WVNPs showed a higher cytotoxic effect compared to BVNPs. Moreover, it was also shown that WI-38 cells required a much higher IC50 concentration regarding all the applied formulations when compared with the cancer cell line, namely HepG2 and MCF-7.
Microscopic examination of HepG2, MCF-7 and WI-38 cells:
After the cytotoxicity assay, the cell lines were morphologically observed by an inverted light microscope to determine any changes evident due to the application of the different toxicants.
The visualization of the changes in cellular density in HepG2 and MCF-7 cell lines after treatment with the different venom formulations (free or loaded on nanogel particles) revealed that the lowest cellular density was evident when free BV was applied, indicating the highest toxicity, followed by free WV, then a moderate cellular density was observed when BVNPs or WVNPs were applied, and the highest cellular density observed was when BNPs were applied.
Statistical analysis:
All data from the cytotoxicity assay were expressed as mean viability ± standard deviation (SD) by Systat SigmaPlot Software, (n=6) where ’n’ represents the number of samples. Moreover, the IC50 was plotted as a regression line and afterwards was fitted by the same software.
The analysis of variance (ANOVA) and Tukey’s multiple comparison test was used to analyze the statistical differences between experimental groups. The lowest acceptable significant threshold, for statistical analysis of all data, was P < 0.05.
Evaluation of the Changes in Expression Levels of NF-κB RELA/p65 in HepG2 and MCF-7 Cell Cultures:
4.1. Total RNA Extraction:
The RNA extraction from the treated HepG2 and MCF-7 cells was performed using the GeneJETTM RNA purification kit (#K0731) according to the manufacturer’s instruction.
Next, RNA concentrations were calculated using the measured absorbance at 260 nm after correcting for the dilution factor used for all samples (dilution factor of 100) pers the following equation:
RNA conc.(µg/ml) = OD260 x 40 x 100
4.2. Removal of genomic DNA from RNA preparations:
Removal of genomic DNA from RNA preparations was done using the DNase I, RNase-free enzyme (#EN0521, Thermo Scientific, USA). The DNase I enzyme was then inactivated by heating.
4.3. First Strand cDNA Synthesis:
The prepared RNA was used as a template for cDNA synthesis using the Thermo Scientific RevertAidTM First Strand cDNA synthesis kit (#K1621), according to the manufacturer’s instruction. The kit uitilizes RevertAid™ M-MuLV (Moloney Murine Leukemia Virus) reverse transcriptase.
Template RNA was mixed with random hexamer primer, reverse transcriptase reaction buffer, RiboLock RNase Inhibitor, dNTP mix and RevertAid M-MuLV reverse transcriptase. This mix was incubated for 5 min at 25oC followed by 60 min at 42oC. The reaction was then terminated by heating at 70oC for 5 min. The product of the first strand cDNA synthesis was stored at -20oC and was later directly used as a template for qRT-PCR.
4.4. Determination of the NF-κB Expression Level by qRT-PCR:
Oligonucleotide primers for real-time polymerase chain reaction (RT-PCR) amplification were ordered from Sigma Scientific Services Co. The β-actin housekeeping gene was employed as an internal standard (a reference gene) for normalization of target gene expression levels, to compensate intra- and inter-kinetic RT-PCR variations (sample-to-sample and run-to-run variations).
The Thermo Scientific Maxima SYBR Green/ROX qPCR master Mix (#K0221) along with the four designed primers (for genes β-actin and NF-κB RELA/p65) were used for the qRT-PCR, and the results were computerized using Stratagene (Mx3000PTM) machine. The master mix included Maxima® Hot Start Taq DNA polymerase and dNTPs in an optimized PCR buffer. It also contained SYBR® Green I dye and was supplemented with ROX passive reference dye.
The master mix was mixed thoroughly and appropriate volumes were dispensed into wells of PCR plates containing template DNA. The reactions were then mixed gently without creating bubbles to avoid any interference with fluorescence detection. Thermal cycling was then performed using a three-step cycling protocol with the fluorescence being measured at the end of each cycle.
All reactions were performed in triplicates to confirm reproducibility, and included a negative control (without template) to verify that no primer–dimers were being generated.
A comparative threshold cycle (∆Ct) value is calculated for each sample as the difference between the Ct (cycle threshold) values for the gene of interest (RELA/p65) and the endogenous housekeeping gene (β-actin) in each sample. The ∆∆ Ct value is the difference between the ∆Ct values of an experimental sample and the control sample. The fold change in gene expression is equal to 2-∆∆Ct.
The fold change expression difference for NF-κB, between the control and treated cells were calculated by normalizing with β-actin gene expression according to the following formula:
Concerning the gene expression of RELA/p65, the qRT-PCR data revealed that the RELA/p65 mRNA expression levels in HepG2 were most down-regulated by free BV (2.09), followed by free WV (2.23), BVNPs (3.16) and WVNPs (3.30). This was evident by depression in the fold change of RELA/p65 NF-κB gene compared with the control β-actin.
Similar results were evident with the MCF-7 cell line, where the RELA/p65 mRNA expression levels were most down-regulated by free BV (2.17), followed by free WV (2.39), BVNPs (3.48) and WVNPs (3.50).
CONCLUSION
The findings of the present study concluded the following:
The formulated chitosan-polyacrylic acid nanogel carrier exhibited a convenient size range and demonstrated efficient encapsulation with both the bee and wasp venoms without indicating any chemical alterations in either its polymeric structure or that of the loaded venoms.
The cytotoxic behavior of the free venoms was considerably higher than that of the nanogel-conjugated venoms, while the bare nanogel showed a very low cytotoxic behavior, indicating its possible safety.
The applied toxicants showed much lower cytotoxicity against normal WI-38 cell line compared to the cancerous HepG2 and MCF-7 cell lines.
The cytotoxic behavior of the free venoms and their nanogel-conjugated forms was further evident in the downregulation of the NF-B RELA/p65 activity.
As a result, the chitosan-polyacrylic acid nanogel particles loaded with bee and wasp venoms are strongly regarded as efficient and non-toxic carriers with possible synergistic and selective effects on the bee and wasp venoms against liver and breast cancers, and are further proposed to be a promising therapeutic approach against liver and breast cancers.