Delivery of Melittin Loaded Niosomes for Breast Cancer Treatment: an in-vitro and in-vivo Evaluation of Anti-cancer Effect

Melittin, a peptide component of honey bee venom, is an appealing candidate for cancer therapy. In the current study, Melittin and Melittin loaded niosome had been optimized and assessed the anticancer effect an In-Vitro on 4T1 and SKBR3 breast cell lines and In-Vivo on BALB/C inbred mice. "Thin-layer hydration method" was used for preparing the Niosomes; different niosomal formulations of Melittin were prepared and characterized in terms of morphology, size, Polydispersity index, encapsulation eciency, release kinetics, and stability. A niosome was formulated and loaded with Melittin as a promising drug carrier system for chemotherapy of the breast cancer cells. Hemolysis, apoptosis, cell cytotoxicity, invasion and migration of selected concentrations of Melittin, and Melittin loaded niosome were evaluated on 4T1 and SKBR3 cells using hemolytic activity assay, ow cytometry, MTT assay, soft agar colony assay, and wound healing assay. Real-time PCR was used to determine the genes expression. 35 BALB/c inbred mice were used; then, the histopathology, P53 immunohistochemical assay and estimate of renal and liver enzyme activity for all groups had been done. Results: This study showed Melittin loaded niosome is an excellent substitute in breast cancer treatment due to enhanced targeting, encapsulation eciency, PDI, and release rate and shows a high anticancer effect on cell lines. The Melittin loaded niosome affect the genes expression by studied cells were higher than other samples; down-regulates the expression of Bcl2, MMP2, and MMP9 genes while they up-regulate the expression of Bax, Caspase3 and Caspase9 genes. Also enhanced the apoptosis rate and inhibitored cell migration, invasion in both cell lines compared to the Melittin samples. Results of histopathology showed reduce mitosis index, invasion and pleomorphism in Melittin loaded niosome. Renal and hepatic biomarker activity did not show a signicant difference in Melittin loaded niosome and Melittin compared to healthy control. In P53 did not show a in all groups. represent mitotic cells and severe cell polymorphisms (a). In healthy control, are any cells and in derma (b) polymorphism (d) Melittin Examination few numbers of tumor very low polymorphisms, fewer inammatory cells (e). Evaluation of breast tissue that treatment with 3 mg/kg Melittin loaded niosome numbers of tumor very low polymorphisms, and fewer inammatory Melittin and Melittin loaded niosome compared to the healthy control group did not show signicant 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.

The Bcl2 protein family, of which Bax is a member, plays a critical role in cell death or survival [17,18]. Bcl2 is expressed in solid tumors, such as breast, colorectal, prostate, stomach, lung, ovarian, and cancers [18]. Bcl2 family proteins expressed in normal mammary tissue [18]. Bcl2-positive expression in breast cancer a sign of estrogen receptor functional activity [19,20]. The balance between Baxas a pro-apoptotic and Bcl2 as a anti-apoptotic protein levels is necessary for the regulation of apoptosis and overexpression of Bax leads to apoptosis in cells, suggesting that tight regulation of Bax, from transcription to post a translation, is important for cell survival [21] .
MMP2 and MMP9 are mainly secreted by tumor cells and stromal cells in the form of zymogens, they play main role in degrading extracellular matrices and metastasis and promoting tumor invasion [22,23]. Melittin has anticancer effects on 4T1 breast cancer cells with up-regulation on Bax, Mfn1, Caspase3 and Caspase9 and down-regulation on Bcl2, Drp1, MMP2 and MMP9 geans so it can be a best candidate for further research on breast cancer treatment [24]. Therefore, combination therapy with more precise technique to maximize the e cacy of the therapy is valuable. Nanoparticles conjugation with chemotherapeutic drugs and natural compounds with anti-cancer effect showed some promising outcome, with many of them approved for treatment of different cancer types [25].
Nanotechnology is an innovative scienti c eld that takes account of the eccentric features at the nanoscale. Nanoparticles provide a high surface area to mass ratio and usually interact e ciently with their surroundings, but they can work as contained carriers for their constituent molecules rather than the same molecules in solution [26]. Therefore, nanoparticles are promising carriers for targeted delivery of therapeutic substitutes. Nanoparticles have been designed for optimizing size and characteristics to magnify the biodistribution of cancer drugs in the bloodstream. They can transfer their loaded active drug to cancer cells by selectively using tumors' speci c stimuli [27,28]. Drug resistance is another obstacle that hinders the e cacy of molecularly targeted and precise chemotherapeutic operators and can reduce nanoparticle applications. Multifunctional and multiplex nanoparticles are now being actively investigated as aiding, personalized, and tailored cancer medication. Drug delivery systems are characterized as formulations aiming to convey a drug to the desired area of action through the body [29]. Niosomes are special drug carriers developed by the self-association of cholesterol and nonionic surfactants in an aqueous phase [27,29]. They are an alternative to phospholipid vesicles to encapsulate hydrophobic and hydrophilic drugs providing su cient encapsulation capability, biocompatibility, biodegradation, low preparation cost, and ample stability [28,29].
The presented study aims for a more precise and effective drug as a cancer remedy with approaching combination treatment, using Melittin and loaded into Nanoniosomes. In this work, the impact of niosomal formulations had been assessed on 4T1 and SKBR3 breast cancer cell line proliferation with free drugs and evaluated its possible effects on Bax, Bcl2, Caspase3, Caspase9, MMP2 and MMP9 mRNA expression and effect on inbred BALB/c mice.

Melittin-loaded Niosomes Characteristics:
The encapsulation e ciency (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 e ciency, 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 e ciency 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 e ciency (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 speci c amount (1mg) of Melittin as a drug molecule, then sonicated for ve 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 uni cation 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.

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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 [32]. It can be concluded from Table 1, adding tween60 to the   formulation represents the zenith encapsulation e cacy 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 e cacy 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].

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 [28]; 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 rm 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.

In-Vitro drug kinetic and release studies of Melittin from Niosomes:
To investigate the In-Vitro drug release, every selected formulation drug release pro le 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 rst had burst in the bloodstream (82.19% during rst 8 hours); after 24hours, it had reached to monotonous release manner for the next hours. Niosomal Melittin release pro le surveillances showed that in 7.4 pH, 43.45% of the drug had penetrated in the rst 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 [35]. 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 [36].
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, rst-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 rst-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 [39].

Physical Stability Study of Niosomal Melittin:
Vesicle size, polydispersity index (PDI), and encapsulation e ciency (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 gure (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 [34]. 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 puri ed Melittin from honey bee venom. The ndings 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 HD 50 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 speci city [40,41].

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. Melittin exempli es 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][44][45][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 [48]. 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 [49].

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.
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 [50]. 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].

Flow cytometric analysis
Apoptosis of breast cancer cells was measured by double staining using annexin V uorescein isothiocyanate (FITC) and propidium iodide (PI). In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is exposed to the external cellular environment Annexin V could be served as a sensitive probe for the ow cytometric analysis of cells undergoing apoptosis due to its high a nity for PS, even when it conjugated with FITC. Furthermore, PI can stain the DNAs in the ow 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. Breast cancer cell invasion and growth of tumor cells can be inhibited by Melittin [10]. 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 [53]. Hesperidin [54,55], Piperine [56], BV and Melittin [57,58] have also shown anti-cancer activities on breast cancer cells.

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 gure, 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 gures 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&

Weight and volume changes
On the day before commencing all animals had same weight (about 19±0.20 gr) and tumor volume (3 mm 3 ) 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
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 identi ed. Subsequently, the mitosis index was evaluated in 10 elds with a magni cation 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 = in ltration 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 in ammatory cells (c). There is tumor mass beneath the skin, indicates necrotic tissue and shows the prominent nuclear polymorphism (d) that was exactly in

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 ndings 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.

Conclusion
The presented study was aiming for adequate, more effective, and less harmful breast cancer treatment. Our study successfully declares that Melittin-loaded nano-Niosome properly functions under the ideal-estimated status. This project has demonstrated that Niosomes are suitable vesicle carriers for combining drugs, particularly Melittin. Melittin loaded Niosome reported had strongly higher anti cancer effects in-vivo and in-vitro than free Melittin. Hopefully, this formulation can be industrialized in the next few years and reduces the breast cancer incidence rate.

Melittin loaded Niosomes Preparation
"Thin-layer hydration method" prepared the drug-loaded Niosomes containing cholesterol, span60 and tween60, reported in our previous work with minor modifications [27,30]. Briefly, surfactants and cholesterol, were dissolved in 10 mL chloroform as organic solvent. A rotary evaporator (120 rpm, 60 °C, one hour) was used to evaporate the chloroform (Heidolph Instruments, Germany). Then, the dried thin films were hydrated utilizing Melittin solution (in PBS, 10 mL, pH 7.4) at 60 °C for one hour (120 rpm). Finally, the sample was sonicated for 5 min (Hielscher up50H ultrasonic processor, Germany, Amplitude: 25%, 200 w) to obtain the niosomal formulations with uniform size distribution ( Table 1). The samples were kept in a refrigerator (4 °C) for further experiments.

Morphology, Size and Polydispersity of Index Measurements
Malvern Zeta Sizer (Malvern Instrument Ltd. Malvern, UK) was applied to distribute the size and polydispersity index based on dynamic light scattering (DLS). To investigate the optimum formulation morphology, transmission electron microscopy (TEM) at 80 KV (Netherland, Philips CM30), scanning electron microscopy (SEM) (SSX-500, Shimadzu, Japan) were utilized.

Determination of Encapsulation E ciency (EE)
The Niosomes were ultra-ltered for 20 min at 4000×g, utilizing an Amicon. During ltration, free drugs moved through the lter membrane, and the Niosomal Melittin remained in the top chamber. Drug concentration at a wavelength of maximum absorbance peak of the drug molecule was analyzed by UV/Visible spectroscopy (JASCO, V-530, Japan), and drug concentration was evaluated according to its standard curve. Finally, the encapsulation e ciency estimated using the following equation:

Encapsulation E ciency (%) = [(A-B)/A] *100
In this equation, "A" is the amount of initial drug trapped into the Niosomal formulations, and "B" stands for non-Niosomal loaded drugs released from the membrane.

In-Vitro Drugs Release Kinetic Study
The In-Vitro Melittin release from Niosomes was analyzed through the following method. Brie y, 2 ml of each sample was added to a dialysis bag. The dialysis bag with each sample was put in PBS solution (pH = 5.4, 6.5, 7.4) and stirred at 37 °C (50rpm). Then Aliquots were taken at speci ed intervals and the medium was refreshed. Several kinetic models were investigated and analyzed the release pro le.

Cells culture
The mouse breast epithelial 4T1 and SKBR3 cell line purchased from Pasteur Cell Bank, Iran. RNA. The cell line was cultured at 37°C and in a 5% CO 2 in air atmosphere. All cells tested negative for mycoplasma contaminations and were markedly cultured in fresh medium (RPMI1640) supplemented with 10% fetal bovine serum (FBS, DENAzist Asia's C o ) and 1% antibiotics (penicillin/streptomycin). The cells (1 × 10 6 cells/ml) were plated in T-25 asks containing 5 mls of RPMI1640 and grown in a humidi ed incubator under an atmosphere of 95% air and 5% CO2 at 37 °C to sub con uence (90 -95%). The culture medium was replaced every 48 hours. Once the cells reached 90 -95% con uency, the medium was aspirated, and the cells monolayer was washed three times with sterile phosphate buffered saline. The cell monolayer was treated with 1 ml of 0.25% (w/v) trypsin-EDTA and incubated brie y at 37 °C and visualized microscopically to ensure complete cell detachment. Cells were re-suspended in complete growth medium. Cells were also stained with trypan blue (100 μl of cell suspension and 100 μl of 0.4% trypan blue), incubated for 2 minutes at room temperature, and counted using a hemocytometer.
2.8 Cell proliferation assay 4T1 and SKBR3 cell lines were separately seeded in a 96-well plate at a density of 5 × 105/well including RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin and 2% L-glutamine. Cells incubated under an atmosphere of 95% air at 37°C and 5% CO2 for 24 h reaching 70-80% con uence. 8, 16, 32, 64, 128 and 256 μg/ml concentrations of Melittin and Melittin loaded niosomes were added and cells were incubated for 48 and 72 h. Incubation of the cells with 0.5 mg/ml of MTT was performed for 4 h followed by replacing the medium with 100 μl of DMSO and vortexing for 20 min. A microplate reader was used to measure the absorbance (570 nm). Median inhibitory concentration (IC50) was calculated for samples.
2.9 Soft agar colony assay 4T1 and SKBR3 cells treated with Melittin and Melittin loaded niosome were suspended in 0.35% agarose and RPMI 1640 supplemented with 20% FBS and seeded over a basal layer of 0.5% agarose. The experiments were established in 100 mm petri dishes at a cell density of 6 × 10 3 cells/well. Colonies were manually counted in nine random elds after 21 days of culture at 37 °C.
Phase contrast micrographs of the colonies were captured with a olympus (Tokyo, Japan).

Wound healing assay
To study cell migration, 4T1 and SKBR3 cancer cells were seeded at 5-105 cells/well in 24-well plates and incubated until they reached 70% con uence. Monolayers were scratched with a 200 μl pipette tip to create a wound, and cells were then washed twice with serum-free culture media to remove oating cells. Media were replaced with fresh serum-free medium. Cells were subjected to the indicated treatment with 8, 16, 32, 64, 128 and 256 μg/ml concentrations of Melittin and Melittin loaded niosomes in medium for 72 hours. Then Cells were washed with PBS, xed and microscopic photos were made.

Flow cytometric analysis
To evaluate the apoptosis/necrosis ratio, the 4T1 and SKBR3 cells were treated with Melittin and Melittin loaded niosomes IC50 concentration for 72 h, and then the cells were studied using Annexin V/propidium iodide (PI) assay, according to the manufacturer's instructions. Brie y, cells were two times washed using cold PBS followed by resuspending in 1X binding buffer (5×105 cells/ well). A culture tube (5 ml) was then lled with 100 μl of the solution. The tubes were then lled with FITC Annexin V and PI (each 5 μl). A gentle vortex was considered followed by incubation (25°C/15 min) in the darkroom. The tubes were provided by 400 μl of 1X binding buffer. Flow cytometry was applied for analysis for 1h. The cells without any treatment were used as control. Finally, the levels of apoptotic/necrotic cells were evaluated using flow cytometry. Centrifuge at 12000 rpm at 40C for 15 min. Transfer the Aqueous phase to a new RNase-free 1.5 ml tube, (do not disturb the midphase) and add an equal volume of isopropanol. Gently mix and incubate ice for 15 min. Centrifuge the mixture at 12000 rpm at 40°C for 15 min. Discard the supernatant and add 1 ml of 75% Ethanol, shortly vortex to dislodge the pellet and then centrifuge at 4 °C for 8 min at 7500 rpm. Discard the supernatant and let the pellet to dry at room temperature for a few minutes (do not let dry completely, it will decrease the solubility of the pellet. Dissolve pellet in 50 μl of DEPC water. To help to dissolve, place the tube in a 55-60 0C water bath for 10 min. Total RNA was extracted using Tripura reagent according to the manufacturer's instruction.

cDNA synthesis
Mix the template RNA (total RNA or Poly (A) mRNA) 1ng˜5μg, Buffer-Mix (2X) 10 μl, Enzyme-Mix 2 μl and DEPC-treated water up to 20 μl components in RNase-free tube. Mix the above mixture by quick vortex then incubate 10 min at 250C. Incubate 60 min at 470C. Stop the reaction by heating at 850C for 5 minutes. Chill on the ice or at 40C. To perform PCR, you can add the nished RT reaction up to 1.5 of the nal PCR volumes.

Primer design and RT-PCR
The particular primers for Drp1, Mfn1, Bax, Bcl2, Caspase3, Caspase9, MMP2, MMP9 and ß-actin (as internal control) were designed through the National Center for Biotechnology Information (NCBI) website (Table 2). In this study 35 females, BALB/c inbred mice (weighing 18±2 g, 6 to 8 weeks) were individually housed in polycarbonate animal cages and maintained under constant temperature (22 ± 2°C) and humidity (55%). The mice had free access to food and water and were kept in 12 h light-dark cycles. After 1 week of acclimatization, the BALB/c mice were randomly divided into seven groups (n = 5 per group).

Induction of Breast cancer
The experimental breast tumor was induced by subcutaneous injection of 4T1 tumor cells (105 /mL in suspension with phosphatebuffered saline, PBS 1X). The injection site was on the right side of the chest pad. 12th day after tumor induction, a solid tumor appeared subcutaneously. After 2 weeks, tumors were palpable. Tumor volume and mice weight was measured by using a digital scale and a digital caliper and calculated according to Formula 1 and formula 2, mentioned below; The end of the study period, animal blood samples were taken then liver and renal biomarkers were measured. The level of activity of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), Albumin, Creatinine, blood urea nitrogen (BUN), Total Protein in the collected samples was determined spectrometrically using "Pars Azmoon kit."

Histopathology
Samples LD50 were considered in treatment groups, daily intraperitoneally (i.p.) injection of samples for 20 days were performed. Three mice of each group were sacri ced at the end of the treatment period. The tumor mass was removed and xed in 10 % buffered formalin. Tumor tissues were processed in a tissue processor, and para n-embedded tissue sections were stained by hematoxylin and Eosin (H&E) method. The tumor was histologically classi ed according to the Nottingham histologic score system (Menten grade) [31]. By this scoring system, the amount of nuclear features, gland formation, and mitotic activity was assessed. All methods used in the study comply with the institutional ethical guidelines for care and the use of animals in research.

Immunohistochemical analysis of the tumors
Tumor tissues were xed in 10% formalin buffer solution, washed three times in PBS, and left in 70% ethanol. Tumors were keeped in para n, and 5-μm sections were prepared. For Hematoxylin and Eosin stainin, slides were dewaxed, hydrated using a decreasing solution bank of ethanol, stained with Gill'sg hematoxylin, dehydrated using 70% ethanol, stained with eosin, further dehydrated using 100% ethanol, cleared using toluene, and mounted in coverslips using Acrymount IHC mounting media (StatLab). Tumor cells apoptosis were determined in tissue sections by TUNEL assay (In Situ Cell Death Detection Kit, Roche).

Statistical analysis
Data were reported as mean ± SD and the graphs were plotted using GraphPad Prism version 8. Data were statistically analyzed using analysis of variances (ANOVA) followed by post-Tukey test and a p-value less than 0.05 was considered as a signi cant difference.

Declarations
Ethics approval and consent to participate There are no "human subjects" in this study Consent for publication Not applicable Availability of data and materials  Figure 1 Morphological determination of optimized formulation. A: Analysis of particle size distribution of Melittin loaded niosome by Dynamic Light Scattering (DLS); B: Scanning electron microscopy (SEM); C: Transmission electron microscopy (TEM). Comparing Stability of optimum formulation at 4°C and 25°C. Mean particle size (a), PDI (b) and EE % (C) were studied as stability parameters. Results are represented by mean ± SD (n = 3). *p < 0.05, ***p < 0.001.     The ow cytometric analysis diagram of Melittin and Melittin loaded niosome provided in SKBR3 (Figure 9a) and 4T1 breast cancer cell lines (Figure 9b).