3.1 Melittin-loaded Niosomes Characteristics:
The encapsulation efficiency (EE) and the size of the niosome much depend on the type of surfactants and the volume of cholesterol (i.e., lipid) in the niosomal structure because any change in the chemical species and chemical formation undeviatingly affects the hydrophilic-lipophilic balance (HLB) in the niosomal formulation. A suitable drug delivery system must have high encapsulation efficiency, a small size, and a structure to carry an ample amount of drug, penetrate the target tissue, and release the drug molecules inside the target tissue. However, there is an obscure association between the size and encapsulation efficiency in the niosomal formulations. Various formulations have been prepared and investigated to optimize the arrangement. Table 1 shows the size, polydispersity index (PDI), and encapsulation efficiency (EE) of the niosomal formulation in terms of the span60 percentage (i.e., in the Span 60/Tween 60 mixture) and cholesterol content for a specific amount (1mg) of Melittin as a drug molecule, then sonicated for five minutes to provide more uniform Niosomes. Melittin has multiple hydrophilic groups (e.g., OH, and NH), which inaugurates an interaction between Melittin and the hydrophilic chain of niosome. Hence, a unification of two surfactants with low and high hydrophilic-hydrophobic balance (HLB) may result in Melittin's tremendous encapsulation with the small size of niosomal formulation. Tween 60 is a nonionic surfactant with a high hydrophilic moiety, while Span 60 is a nonionic surfactant with a hydrophobic fraction.
Consequently, varying Span 60/Tween 60 ratios could accurately regulate the hydrophilic-lipophilic balance (HLB) of the surfactants and directly modify their cooperation with drug molecules . It can be concluded from Table 1, adding tween60 to the formulation represents the zenith encapsulation efficacy and PDI, while it provides the smallest size (Table1, T2, and T5). As shown in Table 1, T1 to T3, when the surfactant/cholesterol ratio was 1:1, the particles' mean size is smaller than when the ratio changed to 2:1, due to a thicker lipid layer, which cholesterol provided. However, applying tween60 to the formulation provided an adequate EE and size; utilizing it separately in cholesterol combination without span60 resulted in a larger size and less EE (Table1, T3, and T6). It was evident from the experiment results that span60 cannot provide either a suitable encapsulation efficacy and particle size (Table2, T1, and T5). All the results conclude that the combination of span60 and tween60 with the cholesterol possesses the optimum drug carrier for hydrophilic drugs like Melittin [33, 34].
3.2 Morphological characterization:
The morphology of Melittin-loaded Niosomes was assessed using SEM and TEM methods. In this study, the Niosomes particle size was less than 50nm, much smaller than DLS observation. This variance can be due to the difference between SEM and DLS techniques ; in SEM, the dried samples are examined, but in DLS, the samples might be hydrated; thus, the particles' size in the DLS test is more prominent. The examination demonstrates a smooth surface, spherical form, and separated firm boundaries with a uniform distribution. The size of nanoparticles has been assessed usingDynamic Light Scattering DLS. As Figure 1 shows, the average diameter is 121.4nm, which represents the optimum formulation size. As it had been mentioned before, other formulations did not provide acceptable sizes for drug delivery applications.
3.3 In-Vitro drug kinetic and release studies of Melittin from Niosomes:
To investigate the In-Vitro drug release, every selected formulation drug release profile was examined for 72 hours in 7.4, 6.5, and 5.4 pH at the body temperature. As it can be seen in the "Release" diagram (Figure 2), the free drug first had burst in the bloodstream (82.19% during first 8 hours); after 24hours, it had reached to monotonous release manner for the next hours. Niosomal Melittin release profile surveillances showed that in 7.4 pH, 43.45% of the drug had penetrated in the first eight hours; this rate increased to 44.91 in 6.5 pH and 51.29% in 5.4 pH. After 72hours, 72.19%, 80.81%, and 92.11% of the drug released into the bloodstream in 5.4pH, 6.5pH, and 7.4pH, respectively, attributed to the acidic condition Niosomes-swelling structure . Niosomes' acidic departure is related to electrophilic addition reactions. The drug-loaded Niosomes had studied for the release rate. Acidic pH crushed the Niosomes structure, increasing the release rate, increasing the toxicity as the tumor wards habitually have the acidic condition . Also, the acidic condition affects the Melittin and increases the osmotic pressure, which induces more cytotoxicity [37, 38]. Melittin's release data had been mathematically measured, in zero-order, first-order, Korsmeyer-Peppas, and Higuchi's orders, in three pH range (7.4, 6.5, and 5.4) for 72 hours in body temperature, Table 3. Free drug release followed the first-order model, with the rate of R2=0.9643, representing a drug-concentration release (this applies for Melittin, as a separate free drug). Melittin-loaded Niosomes had followed the Korsmeyer-Peppas model (n) in either 7.4, 6.5, and 5.4 pH. This fact declared that the release mechanism is the diffusion-erosion arrangement. The release rate in each, 5.4, 6.5, and 7.4 pH, are R2=0.9431 with n=0.4021, R2=0.9297 with n=0.4141, and R2=0.9003 with n=0.4454 respectively .
3.4 Physical Stability Study of Niosomal Melittin:
Vesicle size, polydispersity index (PDI), and encapsulation efficiency (EE) were analyzed by putting them at 4°C and 25°C, and on days 0, 14, 30, and 60 after the preparation to pick the optimal Melittin Niosomal formulations and physical stability. Interestingly, the observations demonstrated that the temperature affected neither the size of particles, PDI, or EE percentage and possesses the minimum size with the mean of 121.4nm, maximum PDI (0.211), and EE (79.32%) on the day the formulation just prepared. As shown in the stability figure (Figure 3), in the following days to day60, the temperature affected all the parameters. Increasing the temperature caused size expansion, more PDI, and EE reduction. The EE reduction is due to the rise of drug release in terms of temperature increase . As the temperature can affect the rigidity and elasticity, growing the pores of the Niosomes; could be effective on the particles size and PDI and increase either of them and reduce the EE to its minimum amount (55.19%). As can be concluded from the stability results, the stability is better at 4°C attributed to the rigidity and elasticity of the niosomes, because at 25°C, the grown pores caused bigger size, more PDI, and less EE.
3.1. Hemolytic Activity of Melittin in BALB/c Mice Erythrocytes
A powerful hemolytic activity was observed in the purified Melittin from honey bee venom. The findings indicated that Melittin had a powerful hemolytic activity at the concentrations of above 0.5 μg/ml (Figure 4). 0.125 μg/ml and 0.25 μg/ml concentrations of Melittin did not indicate considerable lysis effect. The HD50 value obtained 2 μg/ml indicating is the concentration, at which Melittin shows 50% of the hemolytic activity of the positive control.
Melittin is composed amino acid residues and Melittin's hemolytic activity has been demonstrated and It is known to have a powerful hemolytic activity against mammalian and bacterial cells due to its weak cell specificity [40, 41].
3.5 Cell proliferation assay
The treatment of two breast cancer cells (4T1 and SKBR3 cells) with niosomal formulation resulted in a higher inhibitory effect (less cell viability) compared to free drug solutions. To determine the inhibitory effect of individual Melittin as a free form and Melittin loaded niosome as a niosomal form on 4T1 and SKBR3 cells. A dose-response experiment had been performed for both groups. As indicated in individual treatments with the free form and the niosomal form resulted in growth inhibition of 4T1 and SKBR3 cells in a dose-dependent pattern.
IC50 value was evaluated in all study groups on 4T1 mice breast cancer cell line after 48 and 72 h treatment. In study groups (Melittin and Melittin loaded niosome) IC50 values respectively were 143.20 μg/ml and 75.58 μg/ml after 48 h, and 40.62 μg/ml, 27.17 μg/ml after 72h. According to the results in all groups, IC50 remarkably decreased after 72 h compared to 48 h (P<0.001 ***) (Figure 5a). In SKBR3 human breast cancer cell line after 48 and 72 h treatment IC50 value was evaluated. In Melittin and Melittin loaded niosome groups IC50 values respectively were 87.87 μg/ml and 47.65 μg/ml after 48 h and 50.56 μg/ml, 31.05 μg/ml after 72h treatment. According to the results in study groups IC50 remarkably decreased after 72 h compared to 48 h (P<0.001 ***) (Figure 5b). The 4T1 cell line after 48 h of treatment through Melittin loaded niosome (IC50: 75.58 μg/ml) compared to Melittin (IC50: 143.20 μg/ml) IC50 was decrease (P<0.001***). After 72 h of treatment, the results showed the Melittin loaded niosome (IC50: 27.17 μg/ml) compared to Melittin (IC50: 40.62 μg/ml) was decrease (P<0.001***) (Figure 5c). The SKBR3 cell line after 48 h of treatment, the results showed the Melittin loaded niosome (IC50: 47.65 μg/ml) compared to Melittin (IC50: 87.87 μg/ml was decrease (P<0.001***). After 72 h of treatment, the results showed the Melittin loaded niosome (IC50: 31.05 μg/ml) compared to Melittin (IC50: 50.56 μg/ml) was decrease (P<0.001***) (Figure 5d).
The (Figure 6) shows the Niosome and Melittin loaded niosome effects on the MCF10A cell line. The MTT assay was performed for evaluation of cytotoxic effects of different Niosome dilution on MCF10A cells. Niosome dilutions has no significant cytotoxic effects on MCF10A cells separately (Figure 6a). As the Melittin loaded niosome concentration increased, cell viability decreased. So, the 64 and 128 μg/ml compared to control cell viability was decreased (P<0.01** and P<0.001 ***) (Figure 6b). These results indicated that Melittin in free and niosomal forms had more cytotoxicity effect on 4T1 cells and as a model for breast mice mammary epithelial cancer cells compared to SKBR3 cell line after 72h of treatment. The IC50 concentrations were then utilized to generate fixed ratios for subsequent combination experiments and the calculation of combination index (CI).
Melittin exemplifies this large class of membrane-active peptides that manifest membrane-disrupting activity when incorporated into traditional bilayer delivery systems (i.e., liposomes) [42, 43]. Melittin’s action is a physical and chemical disruption of membrane structure resulting in profound compromise of the cell permeability barrier by lysis [43-46]. The peptide partitions into the cell membranes as a monomer, followed by oligomerization into toroidal or barrel stave structures that facilitate pore formation to effect cell death [9, 47]. Melittin have important anti-cancer effects on various types of cancers, including breast cancer so nanocarrier allows accumulation of Melittin in murine tumors In-vivo and a dramatic reduction in tumor growth without any apparent signs of toxicity since nanocarriers selectively delivered Melittin to multiple tumor targets, including cancer cells [9, 47]. In another study the synergistic co-delivery of doxorubicin and Melittin using f nanoparticles for cancer treatment in LC–MS/MS indicated that the co-delivery system of Doxorubicin-Melittin loaded CA-MNPs is highly capable to be used in magnetically targeted cancer therapy . Melittin nano-liposomes would have a better application in hepatocellular carcinoma cells (HCC) therapy due to induced apoptosis in hepatic carcinoma cells In-Vitro and vivo and inhibit hepatocellular carcinoma in LM-3 xenograft tumor model .
3.6 Wound healing assay
Migration is one of the important characteristics of cancer cells and promotes cancer metastasis. To determine the effect of Melittin and Melittin loaded niosome on migration and invasion, In-Vitro wound (scratch) assays were performed in 4T1 and SKBR3 cells and the wound healing rate was monitored through the complete closure of the scratched. As shown in (Figure 7), cell migration on 4T1 and SKBR3 cell lines that treatment with Melittin loaded niosome was lower than Melittin (P<0.001***). 4T1 and SKBR3 cells were investigated using wound healing assay 72 h.
Migration of SKBR3 cell line was decreased by treatment with Melittin loaded niosome with an increase on scratch width (μm) compared to Melittin (P<0.001***) (Figure 7a). 4T1 cell migration was decreased by treatment with Melittin loaded niosome and increase on scratch width (μm) compared to Melittin (P<0.001***) (Figure 7b).
A wound-healing migration assay was showed and demonstrated that Melittin inhibited the vascular endothelial growth factor migration of non-small cell lung cancer cells, when compared with the control group . The development of most human cancers, primary cells move out and invade the neighboring tissues and ultimately travel to distant sites to make new colonies, so targeting cancer cell migration and invasion is an important and necessary aspect of cancer chemotherapy [51, 52].
Soft agar colony assay
To further demonstrate the inhibitory effect of Melittin and Melittin loaded niosome on cancer cells growth, we performed the soft agar colony formation assay. The number of colones was concentration-dependently decreased by Melittin and Melittin loaded niosome compared to control (Figure 8). Number of colones in SKBR3 cell line was decreased by treatment with Melittin loaded niosome compared to Melittin (P<0.001***) (Figure 8a) and in 4T1 cell line the results was same (P<0.001***) (Figure 8b).
3.3. Flow cytometric analysis
Apoptosis of breast cancer cells was measured by double staining using annexin V fluorescein isothiocyanate (FITC) and propidium iodide (PI). In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is exposed to the external cellular environment due to translocation from the inner to the outer surface of the plasma membrane. The flow cytometric analysis diagram of Melittin and Melittin loaded niosome provided in SKBR3 (Figure 9a) and 4T1 breast cancer cell lines (Figure 9b).
To further determine apoptosis induction of 4T1 and SKBR3 cells were stained with Annexin-V/PI assay, followed by flow cytometry, and were compared with cells treated with Melittin and Melittin loaded niosome. The flow cytometric analysis indicated a significant induction in the percentage of apoptosis rate in both groups of cells treated with IC50 of Melittin and Melittin loaded niosome.
Annexin V could be served as a sensitive probe for the flow cytometric analysis of cells undergoing apoptosis due to its high affinity for PS, even when it conjugated with FITC. Furthermore, PI can stain the DNAs in the flow cytometry due to its intercalating property. PI stains the dead cells as it cannot penetrate the membrane of live cells and apoptotic cells. Therefore, it is useful to differentiate necrotic, apoptotic, healthy, and dead cells.
The results demonstrated that the simultaneous administration total apoptosis of Melittin (%), and Melittin loaded niosome (%) in SKBR3 cell line (Figure 9a) and Melittin (%), and Melittin loaded niosome (%) in SKBR3 cell line (Figure 9b) enhances the total apoptosis in both studied cancer cells. These results are in agreement with the cytotoxicity data obtained by MTT assay (Figure 6 a&b).
Breast cancer cell invasion and growth of tumor cells can be inhibited by Melittin . Combination therapy with anticancer drugs has is a well-known approach in cancer treatment. Plant-derived drugs are desired for anticancer treatment as they are secure, accessible and also when are combined with anti-cancer agents, they are able to exert synergistic therapeutic effect, decrease the doses, toxicity and drug resistance of chemotherapeutic agent . Hesperidin [54, 55], Piperine , BV and Melittin [57, 58] have also shown anti-cancer activities on breast cancer cells.
3.7 Gene expression analysis by Real-Time PCR
The inhibitory effect of drugs might be due to the regulation of the expression level of different genes inside the cells. It has been reported that the Melittin and Melittin loaded niosome could affect the expression level of different genes inside the breast cancer cells. Therefore, the expression of eight different genes (Bax, Bcl2, Caspase3, Caspase9, MMP2 and MMP9) inside the two breast cancer cells (4T1 and SKBR3) were measured after treatment of these two cancer cells with different samples containing drug molecules. (Figure 10a) shows the expression levels of Caspase3 gene in SKBR3 cell line treated by Melittin and Melittin loaded niosome. According to the figure, Melittin loaded niosome had higher expression level of Caspase3 than the Melittin group (P<0.001***). (Figure 10b) shows the expression levels of Caspase3 gene in SKBR3 cell line that treated Melittin and Melittin loaded niosome. Caspase9 expression levels in group with Melittin loaded niosome treatment was higher than the Melittin group (P<0.001***). However, in (Figure 10c) Melittin loaded niosome has more Bax expression levels in cells than Melittin (P<0.001***). According to Bcl2 gene expression levels in the SKBR3 cell line that treated by Melittin loaded niosome lower expression level than the Melittin (P<0.01**) (Figure 10d).
On the other hand, the expression levels of Caspase3 (Figure 11a) and Caspase9 (Figure 11b) genes in 4T1 cell line treated by Melittin loaded niosome was higher than Melittin (P<0.001***). Also indicate the mRNA levels of Bax in 4T1 cells treated by Melittin loaded niosome was increased compare to Melittin (P<0.001***) (Figure 11c). The results revealed that Melittin loaded niosome increase Bcl2 expression levels in 4T1 cells compared to Melittin (P<0.001***) (Figure 11d). As shown in figures related to 4T1, the increased MMP2 and MMP9 gene expressions levels can be seen in Melittin loaded niosome comparison with the Melittin (P<0.01***) (Figure 11e & 11f).
According to the (Figures 10 & 11), the expression levels of Caspase3, Caspase9 and Bax in both cell lines, exposed to all study groups were higher than the control group (P<0.001***) and the expression levels of Bcl2, MMP2 and MMP9 in both cell lines, exposed to all study groups were lower than the control group (P<0.001***). As can be seen in (Figure 10& 11), the administration of Melittin loaded niosome shows the synergic and higher up-regulating effects in (Bax, Caspase3 and Caspase9 genes) and down-regulating (Bcl2, MMP2, and MMP9 genes) in both cell lines.
The apoptotic rates of human thyroid cancer cell line (TT) cells were increased following Melittin treatment, Melittin causes increased Caspase3, Caspase9, Bax, and inhibited B-cell lymphoma 2 genes and protein expression .
Melittin decreased the invasion rate of MCF‑7 human breast cancer cell line by down-regulating CD147 and MMP‑9 by inhibiting cyclophilin A expression so the results provide an evidence for Melittin in anticancer therapy and mechanisms .
Honey bee venom contain Melittin and chrysin are effective for destroying chemoresistant ovarian cancer cells through up-regulation of Caspase3 and Caspase9 and down-regulation of Bcl2 genes expression.
3.8 Weight and volume changes
On the day before commencing all animals had same weight (about 19±0.20 gr) and tumor volume (3 mm3) the treatment and last day of the experiment, mice weights and tumor volume showed differences between groups (Table 4). Mice weight showed changes in the Melittin and Melittin loaded niosome groups compare to cancer and healthy controls. According to results, Melittin 6mg/kg and Melittin loaded niosome 3mg/kg control and treatment groups shows increase in mice weight and in compared to all groups the Melittin loaded niosome showed the largest increase. Tumor Volume showed changes in all treatment groups on the last day. The Groups receiving Melittin loaded niosome 3mg/kg, Melittin loaded niosom 1.5mg/kg, Melittin 6mg/kg and Melittin 3mg/kg showed a decrease in tumor volume compared to the control, respectively. Totally, Melittin loaded niosome 3mg/kg showed the greatest effect in inhibiting tumor growth and weight loss in mice.
Histopathology evaluation is
Score of malignancy in the histopathological view according to the results of histopathological studies of nuclear pleomorphism, the following scoring was evaluated: 0 = No pleomorphism / 1 = Small, regular nuclei and a shape / 2 = Moderate degree of difference in size and shape of the nucleus and hyperchromatic of the nucleus with the presence of nuclei / 3 = Severe degree of difference in nucleus size with hyperchromatic nuclei and often with one or more nuclei identified. Subsequently, the mitosis index was evaluated in 10 fields with a magnification of 40 (HPF) and the invasion index of the tumor cells was obtained and the following divisions were obtained:
0 = no mitosis / 1 = 9-1 mitosis / 2 = 10-19 mitosis / 3 = more than 20 mitosis
0 = absence of tumor cells in dermis and hypodermis / 1 = penetration into dermis / 2 = infiltration into hypodermis / 3 = penetration into subcutaneous muscle tissue (Table 5).
In the cancer control group extremely invasive tumor cells in the hypodermis and underlying muscle layer, severe necrosis at the center of the tumor, severe pleomorphism and severe hyperchromasia with high mitosis. The results indicate Necrotic cells, represent mitotic cells and severe cell polymorphisms (a). In healthy control, there are not any cancer cells and tissue in derma (b) and the group, with Melittin 3mg/kg treatment, there are few tumor cells that they are associated with inflammatory cells (c). There is tumor mass beneath the skin, indicates necrotic tissue and shows the prominent nuclear polymorphism (d) that was exactly in line with the results of the group with 3 mg/kg of Melittin group. Examination of mammary gland tissue sections that treated with 1.5mg/ kg of Melittin loaded niosome show a few numbers of tumor cells are found with very low cellular polymorphisms, and fewer inflammatory cells (e). Evaluation of breast tissue sections that treatment with 3 mg/kg Melittin loaded niosome show a few numbers of tumor cells are found in the skin hypodermis with very low cellular polymorphisms, and fewer inflammatory cells (f) (Figure 12).
Evaluated the efficacy of Melittin-loaded nanoparticles in immunocompetent mice. Syngeneic B16F10 mouse melanoma tumors in C57BL/6 mice actively secrete angiogenic growth factors and have already developed a vascular supply . we have demonstrated that synthetic nanoscale vehicles such as nanoparticles can deliver a model cytolytic peptide (Melittin) by flexible passive and active molecular targeting to kill established solid tumors and precancerous lesions .
3.10 Evaluation of renal and liver enzyme activity
Determination of renal serum biomarker including BUN and Createnine the groups treated with Melittin and Melittin loaded niosome compared to the healthy control group did not show significant changes. While the groups treated with Melittin loaded niosome 3mg/kg ratio compared to healthy control group showed higher effect than another groups. In the study of serum liver enzymes (AST, ALT, ALP, Albumin and Total Protein), there are not any changes in all groups compare to control.
Chronic 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding in mice increased the serum levels of AST, ALT, AP and bilirubin. Of particular interest, the study showed that treatment with Melittin appeared to decrease AP and bilirubin concentrations in the serum of DDC-fed mice. Elevations of serum AST, ALT, AP and bilirubin, from liver metabolic disorder, play important roles in the initiation of liver fibrosis, and liver metabolic disorder was affected by pro-inflammatory cytokines that Melittin inhibited these disorders . Melittin is beneficial for the prevention of acute hepatic failure in antitubercular drug-induced hepatoxicity through regulate of AST,ALT, ALP,BUN and Total bilirubin .
3.13 Immunohistochemical assay P53
According to the results of immunohistochemical assay studies, P53 was not expressed as a prognostic factor in mammary gland carcinoma in all groups and was negative (Figure 13). Accumulated P53 in node-negative breast cancer is associated with an aggressive course and a poor prognosis. Our result shows that P53 accumulation cannot predict breast cancer in this study. A larger study is needed to further investigate the role of another factor as an independent prognostic factor in breast cancer. Taken together, these findings present new possibilities for Melittin treatment in tumor therapy. Although further studies are required to understand the detailed mechanisms interactions, the data presented here indicate that Melittin can be used as an anticancer therapy and represents a promising new approach.